La transmission aux semences est l’un des moyens les plus efficaces de survie et de dispersion pour les champignons phytopathogènes. Les semences ainsi contaminées sont altérées dans leur germination et leur viabilité. De ce fait, nous avons cherché à identifier des mécanismes moléculaires qui pourraient être impliqués dans cette capacité de transmission en utilisant le pathosystème modèle Alternaria brassicicola - Arabidopsis thaliana. Pour cela, nous avons analysé la réponse d’A. brassicicola soumis à différentes contraintes in vitro et in vivo :l’exposition à des métabolites de défenses de la famille des Brassicacées (brassinine,camalexine et isothiocyanate) et à des perturbations de la balance hydrique (dessiccation,sorbitol and PEG) ainsi que lors de la colonisation de la semence à partir des siliques. Nous avons montré que la cible probable des phytoalexines indoliques était la mitochondrie avec notamment une altération de la respiration et du potentiel membranaire mitochondrial après une courte exposition. Nos analyses ont aussi révélé que plusieurs protéines de type hydrophilines-like ou en lien avec formation des eisosomes, semblaient être impliquées dans la réponse au stress hydrique. Nous avons également montré que l’expression de la majorité des gènes codant ces protéines était dépendante d’au moins une des trois protéines kinases,AbSch9, AbNik1 and AbHog1. Enfin, nos analyses in planta ont permis d’identifier un mécanisme inattendu, impliquant le remodelage de la chromatine comme élément potentiel,de la régulation de l’expression génique du champignon lors de l’infection. / Seed transmission is one of the most effective means of survival and dispersal for plant pathogenic fungi. The contaminated seeds are altered in their germination and viability. As a result, we have sought to identify molecular mechanisms that could be involved in this transmission capacity using the Alternaria brassicicola - Arabidopsis thaliana pathosystem model. To do this, we analyzed the response of A. brassicicola subjected to different stresses in vitro and in vivo: exposure to defence metabolites of the Brassicaceae family (brassininin, camalexin and isothiocyanate) and to perturbations of the water balance (desiccation, sorbitol and PEG) as well as during seed colonization from silicics. We have shown that the likely target of indolic phytoalexins is mitochondria, including impaired respiration and mitochondrial membrane potential after short exposure. Our analyses also revealed that several hydrophiline-like proteins or proteins related to eisosome formation appeared to be involved in the response to water stress. We have also shown that the expression of the majority of genes encoding these proteins is dependent on at least one of the three protein kinases, AbSch9, AbNik1 and AbHog1. Finally, our in planta analyses identified an unexpected mechanism, involving the remodelling of chromatin as a potential element in regulating the gene expression of the fungus during infection.

Made available in DSpace on 2016-12-23T13:49:04Z (GMT). No. of bitstreams: 1 Paolla Mendes do Vale de Abreu.pdf: 1824788 bytes, checksum: 68f75796e85ff3e4e63667cc51384d6b (MD5) Previous issue date: 2011-03-04 / O mamoeiro (Carica papaya L.) é uma das fruteiras mais cultivadas e o mamão uma das frutas mais consumidas nas regiões tropicais e subtropicais do mundo. O Brasil é o maior produtor mundial de mamão e os estados do Espírito Santo e da Bahia são responsáveis por mais de 70% da área brasileira produtora deste fruto. As doenças, no entanto, constituem os principais fatores limitantes da produção. A meleira do mamoeiro, causada pelo Papaya meleira virus (PMeV), de genoma de RNA fita-dupla (dsRNA), é uma das que ainda não possui uma cultivar resistente. Uma via de resistência às viroses em plantas é ativada por moléculas de dsRNA. Após perceber a presença do dsRNA, a célula inicia uma rota de degradação de moléculas de RNA, que podem ser virais, impedindo, assim, o progresso da infecção. Este trabalho teve como objetivo avaliar a indução de resistência ao PMeV em mudas de mamoeiro utilizando moléculas de dsRNA extraídas do genoma viral. Quatro diferentes tratamentos foram avaliados qualitativa e quantitativamente por meio do diagnóstico molecular do vírus por RT-PCR convencional e RT-PCR em tempo real, respectivamente, em amostras de folha. As mudas de mamoeiro inoculadas apenas com PMeV mostraram intensa infecção pelo vírus logo nos primeiros dias pós-inoculação, enquanto as mudas inoculadas simultaneamente com PMeV e dsRNA do mesmo vírus mostraram uma infecção viral mais atenuada, o que sugere uma redução no sucesso infeccioso pelo vírus causador da meleira. A inoculação combinada em mudas de mamoeiro do PMeV com o dsRNA viral reduziu o progresso da infecção

Made available in DSpace on 2015-03-26T13:58:38Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1233563 bytes, checksum: f3c5e8276eb2268f4aba38e2e851876e (MD5) Previous issue date: 2013-12-18 / The Brazilian agribusiness sector has been demanding more agile and efficient policies and public services in order to increase its competitiveness in a globalized economy. Confronted by this challenge, the Ministry of Agriculture, Livestock and Foodsupply - MAPA has been trying to innovate and continually improve the quality of its processes and services in order to respond to the growing demands of the productive sector and of the Brazilian society. In the laboratory field, MAPA has been demanding that its official and approved laboratories implement quality management systems in conformity with the General requirements for the Brazilian National Institute of Metrology, Quality and Technology (INMETRO). In this process, method validation is a key technical requirement since it can directly affect the quality of the analytical results. The lack of guidelines which indicate the main parameters to be checked in the validation of plant diagnostic methods by real-time PCR (qPCR) and the lack of consensus in the scientific literature have been compromising proper validations of this kind of method. The qPCR is a molecular biology technique which has been extensively used in the detection and identification of plant pathogens due to its rapidity, selectivity, sensibility, broad dynamic range and automation capabilities. However, some factors may impact the quality of qPCR results, such as: DNA quality, PCR inhibitors, primer and probe quality, etc. Thus, some countries and trade blocs have been regulating the implementation of the ISO/IEC 17025 standard and the validation of molecular diagnostic methods for the detection and identification of plant pathogens. The main parameters to be checked in the validation of PCR methods are: sensibility, selectivity, specificity, repeatability and reproducibility. / O setor agropecuário brasileiro vem exigindo políticas e serviços públicos cada vez mais ágeis e eficazes a fim de aumentar sua competitividade em uma economia globalizada. Diante desse desafio, o Ministério da Agricultura, Pecuária e Abastecimento - MAPA vem buscando inovar e aprimorar continuamente a qualidade dos seus processos e serviços a fim de atender satisfatoriamente as crescentes demandas do setor produtivo e da sociedade brasileira. Dentro da área laboratorial do MAPA, o aprimoramento dos processos e serviços prestados foi acelerado com a exigência da implantação de sistemas de gestão da qualidade competência de laboratórios de ensaio e calibra credenciados, bem como a acreditação dos mesmos junto ao INMETRO. Nesse processo, a validação de métodos de ensaio é um requisito técnico importante uma vez que pode afetar diretamente a qualidade dos resultados. A falta de diretrizes que indiquem os principais fatores a serem verificados nas validações de métodos de diagnóstico fitossanitário por PCR em tempo-real (qPCR) juntamente com a falta de padronização das publicações científicas que tratam do tema vêm dificultando a validação adequada desse tipo de método. A qPCR é uma técnica de biologia molecular que vem sendo largamente utilizada na detecção e identificação de diversos fitopatógenos por sua rapidez, seletividade, sensibilidade, ampla faixa dinâmica de quantificação e possibilidade de automação das análises. No entanto, alguns fatores podem impactar negativamente a qualidade dos ensaios, tais como: a qualidade do DNA extraído, a presença de inibidores de PCR nas amostras, a qualidade dos iniciadores e sondas utilizados, dentre outros. Por esse motivo, alguns países e blocos econômicos têm regulado a implantação da norma ISO/IEC 17025 e a validação de métodos de detecção e identificação de fitopatógenos por técnicas moleculares. Dentre os principais parâmetros a serem observados nas validações deviii métodos de PCR destacam-se: repetibilidade e reprodutibilidade. sensibilidade, seletividade, especificidade,repetibilidade e reprodutibilidade.

Made available in DSpace on 2014-06-11T19:29:44Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-07-18Bitstream added on 2014-06-13T20:19:58Z : No. of bitstreams: 1 souza_ec_me_ilha.pdf: 505202 bytes, checksum: b2e8a6c8e22a5805a00f3381be5d40df (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O mal-do-fio (ou queima-do-fio) é uma doença causada pelo fungo Basidiomiceto Ceratobasidium sp., que afeta diversas plantas frutíferas nativas ou cultivadas. A doença ocorre com mais freqüência em zonas de alta precipitação e temperaturas elevadas, típicas de regiões de florestas tropicais como a Amazônia e a Mata Atlântica. Em São Paulo, recentemente detectou-se a ocorrência do mal-do-fio, em caquizeiro na região de Mogi das Cruzes. Essa doença pode- se tornar importante com a expansão do cultivo de fruteiras no Estado. A maioria das pesquisas sobre o patossistema focalizou a epidemiologia e o controle do fungo. Entretanto a etiologia do patógeno ainda não está totalmente definida, especialmente para populações do fungo infectando caqui e chá no Estado de São Paulo. Há também pouca informação sobre a divergência genética entre populações do patógeno de hospedeiros distintos como caqui e chá. O primeiro objetivo deste trabalho foi determinar o posicionamento filogenético global de populações de Ceratobasidium sp. do caqui e do chá, em relação a espécies de Ceratobasidium sp. descritas no mundo. Foram analisadas seqüências de DNA da região ITS-5.8S do rDNA, inferindo-se a história dos alelos ou haplótipos deste lócus, por meio de métodos filogenéticos, cladísticos e coalescentes. Observou-se que uso de C. noxium é apropriado para denominar espécies associadas ao mal-do- fio em caqui e chá, apesar de C. noxium do caqui e do chá constituírem populações filogeneticamente independentes, as quais denominamos de Grupo Diospyrus e Grupo Camellia. Este estudo trouxe uma contribuição importante para a compreensão das relações filogenéticas e biologia de populações de C. noxium em caqui e chá. Uma vez esclarecidas as questões filogenéticas, o segundo objetivo deste trabalho foi testar a patogenicidade cruzada de... / The white-thread blight is a disease caused by the Basidiomycete fungus Ceratobasidium sp. that affects several native or cropped tree fruits. This disease frequently occurs in zones of high precipitation and high temperatures typical of the tropical forest regions such as the Amazon and the Atlantic Forest. In São Paulo, the occurrence of the white-thread blight was detected only recently on kaki orchards closer to Mogi das Cruzes. That disease can become important with the expansion of the fruit trees cropping areas in the State. Most of the researches about the pathosystem has focused on the epidemiology and control of fungus. However the etiology of the pathogen is not totally defined yet, especially for the fungus populations infecting kaki and tea in São Paulo State. There is also little information available about the biological and genetic divergence between pathogen populations from distinct hosts, such as kaki and tea. The first objective of this research was to determine the global phylogenetic placement of populations of Ceratobasidium from kaki and tea, considering the species of Ceratobasidium described throughout the world. Sequences of the ITS-5.8S region of the rDNA were analyzed, inferring the alleles or haplotypes history for this locus, by phylogenetics, cladistisc and coalescent methods. We observed that the use of C. noxium is appropriate to denominate the fungus species associate with the white-thread blight on kaki and tea, despite the fact that C. noxium from kaki and tea constitutes phylogenetically independent populations, which we denominate Diospyrus and Camellia groups. This study brought an important contribution for the understanding of the phylogenetics and population biology of C. noxium infecting kaki and tea. Once the phylogenetics subjects have been cleared, the second objective of this work was to test the cross-pathogenicity... (Complete abstract, access electronic address below)

A leprose é uma das principais doenças na citricultura brasileira devido à sua ocorrência difundida nos pomares e aos altos custos para o controle químico do ácaro vetor. A doença compromete a produção da planta e sua vida útil, manifestando-se através de lesões locais cloróticas ou necróticas em folhas, ramos e frutos, levando à queda prematura destes órgãos e à seca de ramos. O patógeno, Citrus leprosis virus C (CiLV-C), recentemente classificado como espécie tipo de um novo gênero de vírus de planta, Cilevirus, é transmitido pelo ácaro Brevipalpus phoenicis (Geijskes). Apesar de haver consenso de que a doença tem etiologia viral, ainda existem muitas questões pendentes sobre as interações vírus-planta-vetor, cujas soluções contribuirão para o controle integrado da doença. O presente trabalho teve como objetivo obter informações sobre a interação do vírus com células hospedeiras através de ensaios de imunolocalização das proteínas MP (putativa proteína de movimento), helicase (associada à replicação) e p29 (putativa proteína capsidial) do CiLV-C. Para tal, as sequências codificadoras das ORFs mp e hel foram amplificadas via RT-PCR e clonadas em vetor de expressão. Em seguida, promoveu-se a expressão in vitro das respectivas proteínas em células de E. coli e purificação por cromatografia de afinidade e troca iônica. A proteína MP pura foi utilizada para produção de antissoro policlonal específico que foi testado quanto à especificidade por métodos sorológicos. Os resultados do ELISA mostraram que o antissoro apresentou reação positiva com extratos foliares de lesões lepróticas em todos os estágios de desenvolvimento da doença, quando utilizado em altas concentrações. Além disso, lesões maduras reagiram mais intensamente que lesões mais novas. Por Western Blot, detectou-se somente a proteína pura, não sendo possível obter reação positiva em extrato de lesões foliares. Também não foi possível detectar a MP por imunolocalização in situ. Os resultados em conjunto sugerem que ocorre baixo nível de expressão da MP nos tecidos do hospedeiro. Empregando-se anticorpo policlonal contra proteína p29 do CiLV-C, foi possível detectar o CiLV-C em extratos de lesões foliares de leprose por ambos os métodos sorológicos testados e também por Tissue Blotting. Ensaios de imunolocalização in situ permitiram confirmar que as partículas baciliformes presentes em cisternas do retículo endoplasmático de tecidos de lesões lepróticas em plantas representam de fato vírions do CiLV-C. Além disso, também demonstrou-se que o viroplasma que ocorre no citoplasma representa o sítio de acúmulo da proteína p29. Este mesmo ensaio revelou que partículas baciliformes que ocorrem entre membranas de células adjacentes (intestino médio, glândulas prosomais, músculos e epiderme) de B. phoenicis virulíferos são de fato do CiLV-C. A ausência de viroplasma no ácaro sugere que a relação vírus/vetor seria do tipo circulativo, sem replicação. Baseado neste fato discutem-se alternativas para explicar como o vírus trafegaria do lúmen do intestino até o duto salivar para causar infecção numa planta sadia. / Citrus leprosis is one of the most important diseases in the Brazilian citrus production due to the wide occurrence in orchards and also to the high costs involved in the chemical control of the mite vector. The disease affects the plant production and longevity and it is characterized by localized chlorotic and/or necrotic spots on the leaves, stems and fruits. Affected leaves and fruits may drop prematurely and dieback can be observed in stems. The pathogen, Citrus leprosis virus C (CiLV-C), recently considered as the type member of a new genus, Cilevirus, is transmitted by the mite Brevipalpus phoenicis Geijskes. Despite the consensus that citrus leprosis has viral etiology, there are many pending questions regarding the viral interactions with the infected plant and the viruliferous mites. The solution of these questions may contribute to a better disease integrated management. This work aimed to obtain a better understanding about the virus-plant-vector relationship with the host cell by immunolocalization assays of the putative movement protein (MP), helicase (protein involved in the viral replication) and p29 (putative coat protein) of the CiLV-C. ORFs coding sequences of mp and hel was amplified by RT-PCR and cloned in the expression vector. Afterwards, in vitro expression of these proteins in E. coli and its purification by affinity chromatography were realized. The purified MP was used to produce specific polyclonal antibody that was tested for specificity by serological methods. The ELISA results showed that high concentration antibody reacted with the leaves extracts from lesions in all the disease stage of development. Furthermore, the old lesions reacted more intensely than the younger. Western blot (which detected only the pure protein) and in situ immunolocalization assays failed to detect the native MP in lesioned leave extracts. The results as a hole suggest the occurrence of low expression of MP in host tissue. The polyclonal antibody against p29 was able to detect the virus in lesioned plant extracts by PTA-ELISA, Western Blot, and Tissue Blotting. The viral nature of the putative viral particles, present within endoplasmic reticulum cisternae of infected leaf tissue, was confirmed by immunogold label. The labeling also occurred intensely in the viroplasmas, indicating that these structures represent p29 protein accumulation site. Putative virus particles, visualized in viruliferous B. phoenicis, between membranes of adjacent cells (midgut, prosomal glands, epidermis, muscles), was also immunogold labeled indicating that they represent CiLV-C. The absence of viroplasma in the mite tissues suggests that CiLV-C / B. phoenicis relationship is of the circulative type, without replication. Based on this finding, we search for possible alternatives for the viral circulation in the mite body from the midgut lumen to the salivary duct for the infection of a healthy plant to occur.

Nous avons étudié la voie MAPK Mps1/Slt2 du champignon pathogène du riz Magnaporthe Oryzae. Chez la levure, Slt2 active les facteurs de transcription Rlm1, Swi4 et Swi6. Nous avons identifié le gène orthologue de ScSWI4 chez M. oryzae et étudié les rôles de Mps1, Rlm1, Swi4 et Swi6 en comparant les phénotypes de leurs mutants nuls. Δmps1, Δswi4, Δswi6 ont le même défaut de mélanisation, tandis que Δmps1 et Δrlm1 sont déficients pour la sporulation. L'absence d'hyphes aériens et d'hydrophobicité du mycélium sont des phénotypes spécifiques de Δmps1, tandis qu'une réduction de croissance est associée spécifiquement à Δswi4 et Δswi6. Aucun de ces mutants ne présente une hypersensibilité aux enzymes de dégradation de la paroi (EDPs) et aux inhibiteurs de la paroi (aculeacine A, nikkomycin Z, calcofluor white : CFW). La pathogénie de Δswi4 est réduite, tandis que celle de Δswi6 est similaire à la souche sauvage. Δmps1 et Δrlm1 sont non pathogènes. La transcriptomique comparative de la souche sauvage, Δmps1, Δrlm1 et Δswi4 montre qu'il n'existe qu'un petit nombre de gènes sur- ou sous- exprimés chez ces mutants, le nombre maximal étant observée entre Δmps1 et Δswi4. Le gène AGS1 encodant l'alpha-1, 3-glucane synthase, une cible supposée de Mps1, n'est ni sur- ni sous-exprimé chez ces mutants. La souche sauvage et Δmps1 ont la même sensibilité aux EDPs et au CFW à pH6. Cependant, à pH5, la souche sauvage devient résistante aux EDPs et au CFW à pH6. Cependant, à pH5, la souche sauvage devient résistante aux EDPs et au CFW, tandis que Δmps1 perd cette résistance, suggérant qu'elle nécessite Mps1. Notre étude montre que la voie MAPK Mps1 joue un rôle important dans plusieurs processus biologiques de M. oryzae et précise quels sont les cibles / We have studied the Mps1/Slt2 MAPK signalling pathway in the rice blast fungus Magnaporthe oryzae. In yeast, Slt2 activates the transcription factors Rlm1, Swi4 and Swi6. We have identified the M. oryzae gene orthologous to ScSWI4 and studied the roles of Mps1, Rlm1, Swi4 and Swi6 in M. oryzae by comparing the phenotypes of their null mutants. Δmps1, Δswi4, Δswi6 displayed the same defects in melanisation, while Δmps1 and Δrlm1 are defective in sporulation. Loss of aerial hyphae formation and mycelium hydrophobicity are phenotypes specific of Δmps1, while reduced growth is a specific phenotype of Δswi4 and Δswi6. None of these mutants displayed an increased sensitivity against cell wall degrading enzymes (CWDEs) and cell wall inhibitors (aculeacine A, nikkomycin Z, calcofluor white : CFW). Pathogenicity was reduced in Δswi4, while Δswi6 was as pathogenic as WT. Δmps1 and Δrlm1 were non-pathogenic. Comparative transcriptomic of WT, Δmps1, Δrlm1 and Δswi4 highlighted only a limited number of genes up- and down-regulated in these mutants, with the largest number observed between Δmps1 and Δswi4. The alpha-1,3-glucan synthase encoding gene AGS1, a suggested Mps1 target, was not up- nor down-regulated in these mutants. WT and Δmps1 have a similar sensitivity to CWDE and CFW at pH6. However, at pH5, WT displays a resistance to CWDEs and CFW, while Δmps1 loses this pH5 induced resistance, suggesting it requires the Mps1 pathway. This work shows that Mps1 MAPK pathway has an important role in different M. oryzae biological processes and provides new insights on its transcriptional targets

Magnaporthe grisea est responsable de la pyriculariose du riz, principale maladie de cette céréale. L’entrée du champignon dans la plante hôte se fait via l’appressorium. La différenciation de cette structure résulte d’une réorientation génétique et métabolique, et nécessite une régulation génétique fine. Une étude transcriptomique comparant les stades mycélium et appressorium a permis de montrer qu’environ 1300 ORFs sont surexprimées au stade appressorial. Ce transcriptome a permis l’identification de 32 gènes codant pour des facteurs de transcription pour lesquels dix mutants de délétion ont été générés et caractérisés. L’étude de leur pouvoir infectieux a révélé que le mutant délété du gène TF7 présente une pathogénie réduite de 70% sur plant d’orge. De plus, ce mutant est incapable de former des appressoria sur membrane artificielle sauf en présence d’un inducteur chimique (1,16- hexadecanediol). Par ailleurs, lorsque les appressoria sont formés, ils éclatent au bout de 14 heures. Cette altération peut être compensée par l’addition de sorbitol comme osmoprotecteur. Ce mutant est hypersensible à la Nikkomycine Z, un inhibiteur de la chitine synthase suggérant une altération du métabolisme pariétal. Un transcriptome différentiel réalisé à partir d’appressoria sauvages et mutés différenciés sur membrane de Téflon a révélé que des gènes impliqués dans le métabolisme de la chitine sont sous-exprimés dans le mutant ΔTf7. Le facteur de transcription Tf22 dont la délétion conduit à une réduction de 70% de la pathogénie sur riz a également fait l’objet d’une attention particulière. En effet, la recherche d’homologie a montré la présence de deux protéines homologues à Tf22 chez les deux champignons phytopathogènes S. nodorum et C. nicotianae et au-delà de la conservation d’un cluster potentiel de gènes du métabolisme secondaire, identifié chez C.nicotianae. La caractérisation du mutant a montré que l’expression de ce cluster potentiel est régulée négativement par le gène TF22 / Magnaporthe grisea is responsible for rice blast, the major disease of rice. The entry of the fungus in the host plant is via a specialized cell called appressorium. The differentiation of this structure results from a genetic and metabolic shift, and requires fine control mechanisms. A transcriptomic study comparing vegetative mycelium and appressorium mature stage, characteristic of the pre-penetration step was realized. In order to identify new regulatory networks specific of the appressorial differentiation, we focused on 32 genes encodi. Ten deletion mutants of transcription factor genes were generated and characterized. The study of their infectivity revealed that the TF7 gene deleted mutant has a reduced pathogenicity of 70% on barley plant resulting from an inability to penetrate the plant surface. Moreover, unlike the parental strain, this mutant is unable to form appressoria on artificial membrane except in the presence of a chemical inducer (1.16-hexadecanediol). Moreover, when appressoria are formed, they burst after 14 hours. This alteration can be compensated by a sorbitol solution acting as an osmoprotectant. This mutant is hypersensitive to nikkomycin Z, a chitin synthase inhibitor suggesting an alteration of parietal metabolism. A differential transcriptome was conducted comparing wild and mutated appressoria differentiated on Teflon membrane revealed that genes involved in chitin metabolism are dependent on the transcription factor Tf7. A second transcription factor Tf22 whose, deletion leads to a reduction of 70% of pathogenesis on rice, has also been studied. Indeed, the homology search showed the presence of two proteins homologous to Tf22 for S. nodorum and C. nicotianae. Beyond the conservation of the transcription factor, we observed the conservation of a potential cluster of genes of secondary metabolism identified in C.nicotianae. The characterization of the mutant revealed that expression of this potential cluster is negatively regulated by the gene TF22

Orientador: Ione Salgado / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-22T08:31:31Z (GMT). No. of bitstreams: 1 Vitor_SimoneCespedes_M.pdf: 2469178 bytes, checksum: 5b012d0f044b319a6fb8e2dc382c8242 (MD5) Previous issue date: 2013 / Resumo: O NO é uma molécula sinalizadora versátil muito importante em diversos processos em plantas. Uma de suas principais atuações é na sinalização celular durante o processo de defesa contra o ataque de patógenos. Plantas de Arabidopsis thaliana do genótipo mutante nia1 nia2, deficientes para os dois genes estruturais que codificam para a enzima nitrato redutase (NR), são susceptíveis à bactéria Pseudomonas syringae. Foi sugerido que a resposta de defesa prejudicada no mutante NR-deficiente seria resultante de seus reduzidos níveis de NO, quando comparados àqueles do genótipo selvagem. Em um trabalho recente de nosso grupo, empregando microarranjo de DNA, foi observado que a fumigação com gás NO no mutante nia1 nia2 foi capaz de modular diversos genes relacionados à defesa, alguns dos quais não previamente documentados como responsivos a esse radical. Neste trabalho se analisou por PCR em tempo real o efeito do gás NO na expressão de genes relacionados à defesa em plantas selvagem e no mutante nia1 nia2 infectados com uma linhagem avirulenta da bactéria P. syringae. Genes de defesa, como PR1, foram induzidos pela bactéria e a expressão destes foi maior no genótipo selvagem quando comparado ao nia1 nia2, o que é consistente com a susceptibilidade do mutante. A fumigação com NO também modulou genes relacionados à biossíntese de lignina (CAD1) e à sinalização de auxina (TIR1, ILL1, GH3) e etileno (ACCS7). Análises de quantificação de lignina mostraram uma pequena redução desse composto no genótipo mutante em relação ao selvagem, além de uma diferença em sua composição. Ademais, a fumigação com NO atenuou a expressão de PR1 e outros genes relacionados à via do ácido salicílico em plantas inoculadas e preveniu o crescimento bacteriano em folhas nia1 nia2. Já foi demonstrado que a inoculação do patógeno no mutante induz uma baixa produção de NO e no presente trabalho observou-se uma alta produção de H2O2 comparado ao selvagem. O H2O2 potencializou o efeito microbicida do NO fumigado na suspensão de bactéria. Os resultados sugerem que um efeito microbicida direto do NO, em conjunto com H2O2, pode resultar na atenuação da resposta de defesa na planta, reduzindo o gasto de energia associado à transcrição de genes relacionados à defesa / Abstract: NO is an important signaling and versatile molecule which plays important roles in many processes in plants. One of its main actions is in cell signaling during defense response against pathogen attack. Arabidopsis thaliana plants of the nia1 nia2 mutant genotype, deficient in the two structural genes encoding for the enzyme nitrate reductase (NR), are susceptible to the avirulent bacteria Pseudomonas syringae. It has been suggested that the impaired defense response in the NR-deficient mutant would result from their low NO levels when compared to those of the wild type. Indeed, in a recent study from our group, it was observed through a DNA microarray that fumigation of nia1 nia2 mutant with NO gas was able to modulate many genes related to defense, some of which not previously documented as responsive to this radical. In this work we analyzed by real-time PCR the effect of NO gas on the expression of genes related to defense in the wild type and nia1 nia2 mutant infected with an avirulent strain of Pseudomonas syringae. Defense genes such as PR1 were induced by the bacteria and its expression was higher in wild type when compared to nia1 nia2, which is consistent with the susceptibility of the mutant. NO fumigation also modulated genes related to the biosynthesis of lignin (CAD1) and the auxin (Tir1, ILL1, GH3) and ethylene pathways (ACCS7). Analysis of lignin showed a reduction of this compound in the mutant genotype compared to wild type, and a difference in its composition. In addition, fumigation with NO attenuated the expression of PR1 and other genes related to salicylic acid signaling in infected plants and prevented bacterial growth in nia1 nia2 leaves. Furthermore, pathogen infection is known to induce a low production of NO in nia1 nia2 and here we also observed that there is a higher production of H2O2 in the mutant compared to the wild type. H2O2 potentiated the microbicidal effect of NO fumigated in bacterial suspensions. The results suggest that a direct microbicidal effect of NO, together with H2O2, may result in attenuation of the defense response in the plant, reducing energy expenditure associated with the transcription of genes related to defense / Mestrado / Biologia Vegetal / Mestra em Biologia Vegetal

Plants and microorganisms interact with each other constantly, with some interactions being mutually beneficial and others being detrimental to the plants. The features of the organisms involved in such interactions will determine the characteristics of individual pathosystems. Plants respond readily to pathogen attacks, regardless of the pathosystem; furthermore, variation in the resistance to pathogens within species is common and well documented in many plant species. The variability in pathogen resistance is at the core of genetic improvement programs for disease resistance. True resistance to pathogens in plants is a genetically determined and complex trait that can involve both constitutive and induced mechanisms at different levels of organization. The complexity of this phenomenon makes the study of compatible plant - pathogen interactions challenging, and typically, disease resistance studies focus on specific aspects of a pathosystem, such as field resistance, anatomical or physiological features of resistant plants, or molecular mechanisms of resistance. The Thuja sp. - Didymascella thujina (E.J. Durand) Maire interaction is an important pathosystem in western North America, which has been studied for more than five decades. Western redcedar (Thuja plicata Donn ex D. Don) is very susceptible to cedar leaf blight (D. thujina), a biotroph that affects the tree at all stages, although seedlings are the most sensitive to the pathogen. The characteristics of the Thuja sp. - D. thujina interaction, the wealth of information on the pathosystem and the excellent Thuja sp. genetic resources available from the British Columbia Ministry of Forests, Lands, Natural Resource Operations and Rural Development make this interaction an ideal system to advance the study of disease resistance mechanisms in conifers. This Doctoral project presents a comprehensive investigation of the constitutive and induced resistance mechanisms against D. thujina in T. plicata, Thuja standishii (Gord.) Carrière and a Thuja standishii x plicata hybrid at the phenotypic and gene expression levels, undertaken with the objective of exploring the resistance mechanisms against the biotroph in these conifers. The project also aimed to establish base knowledge for the future development of markers for marker-assisted breeding of T. plicata. The investigations included a combination of histological, chemical and next generation sequencing (NGS) methodologies. NGS data were analyzed, in addition to the traditional clustering analyses, with cutting edge machine learning methods, including grade of membership analysis, dynamic topic modelling and stability selection analysis. The studies were progressively more controlled to narrow the focus on the resistance mechanisms to D. thujina in Thuja sp. Histological characteristics related to D. thujina resistance in Thuja sp. were studied first, along with the relationship between climate of origin and disease resistance. The virulence of D. thujina was also documented early in this project. Chemical and gene expression constitutive and induced responses to D. thujina infection in T. plicata seedlings were studied next. T. plicata clonal lines were then comprehensively studied to shed light on the mechanisms behind known physiologically determined resistance. A holistic investigation of the resistance mechanisms to D. thujina in T. standishii, T. plicata and a T. standishii x plicata hybrid explored the possibility of a gene-for-gene resistance model. Thirty-five T. plicata families were screened during the four field seasons carried out between 2012 and 2015, totalling more than 1,400 seedlings scored for D. thujina severity. Thirteen of those families were used in the five studies performed during the program, along with two T. plicata seedling lines self-pollinated for five generations and three T. plicata clonal lines. One T. standishii clonal line, and one T. standishii x plicata clone were also investigated during the program. A total of 16 histological and anatomical characteristics were studied in more than 750 samples, and more than 270 foliar samples were analyzed for 60 chemical and nutritional compounds. Almost one million transcriptomic sequences in four individually assembled reference transcriptomes were examined during the program. The results of the project support the variability in the resistance to D. thujina in T. plicata, as well as the higher resistance to the pathogen in plants originating from cooler and wetter environments. The data collected also depicted the existence of age-related resistance in T. plicata, and confirmed the full resistance to the disease in T. standishii. Western redcedar plants resistant and susceptible to D. thujina showed constitutive differences at the phenotypic and gene expression levels. Resistant T. plicata seedlings had thicker cuticles, constitutively higher concentrations of sabinene, alpha-thujene, and higher levels of expression of NBS-LRR disease resistance proteins. Resistant clones of T. plicata and T. standishii had higher expression levels of bark storage proteins and of dirigent proteins. Plants from all ages, species and resistance classes studied that were infected with D. thujina showed the accumulation of aluminum in the foliage, and increased levels of sequences involved in cell wall reinforcement. Additional responses to D. thujina infection in T. plicata seedlings included the downregulation of some secondary metabolic pathways, whereas pathogenesis-related proteins were upregulated in clonal lines of T. plicata. The comprehensive approach used here to study the Thuja sp. - D. thujina pathosystem could be applied to other compatible plant-pathogen interactions. / Graduate / 2020-08-31

L'architecture du couvert constitue un levier susceptible de limiter le développement épidémique des mycoses aériennes des plantes. La grande variabilité des caractéristiques architecturales du pois fait du pathosystème Mycosphaerella pinodes/pois un candidat idéal pour une telle étude. Deux hypothèses sont testées pour expliquer la montée de la maladie de la base vers le haut du couvert en cours de culture : i) la présence d'un gradient de réceptivité des organes du pois liée à leur niveau de sénescence, et ii) la présence d'un gradient d'humectation avec une durée d'humectation plus longue à la base des couverts. Au champ, trois cultivars ont été semés à plusieurs densités afin d'obtenir divers scénarios architecturaux. Les couverts les plus denses présentent des niveaux de sénescence plus élevés générés par les indices de surface foliaire des étages supérieurs ainsi qu'un niveau de maladie plus sévère. Une étude analytique complémentaire, réalisée en conditions contrôlées, a montré la plus grande réceptivité à l'ascochytose des organes sénescents. Les mesures microclimatiques montrent une augmentation générale de la durée d'humectation au sein des couverts par rapport à l'extérieur durant les périodes pluvieuses, seules périodes favorables à l'infection d'après notre modélisation adaptée du modèle de Magarey et al. Nos résultats montrent ainsi que l'architecture impacte directement et indirectement le développement épidémique, mais ne peut fournir seule un échappement total à la maladie ; elle doit donc être combinée à d'autres méthodes de lutte.