<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)

Orientador: Suzete Aparecida Lanza Destéfano / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-24T19:29:47Z (GMT). No. of bitstreams: 1 Goulart_MarcelaCristina_M.pdf: 1843141 bytes, checksum: 317126844277b642b5edc69d405b000b (MD5) Previous issue date: 2014 / Resumo: A soja é considerada uma das culturas mais importantes no Brasil, em função de seu alto valor sócio-econômico, determinado pelas inúmeras aplicações de seus produtos e subprodutos e consequente expressão no mercado interno e externo. No entanto, a cultura desta oleaginosa é frequentemente ameaçada com a ocorrência de um vasto número de doenças, que podem acarretar depreciação do produto, redução no rendimento e perdas econômicas para os produtores. Dentre as principais doenças bacterianas que afetam a cultura da soja, destacam-se a pústula bacteriana, causada por Xanthomonas axonopodis pv. glycines (Xag); o crestamento bacteriano, causado por Pseudomonas savastanoi pv. glycines (Psg); e a murcha de Curtobacterium causada por Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), ocasionando perdas na produção de até 40%. A condição sanitária das sementes é extremamente importante se considerarmos que elas são veículos desses agentes fitopatogênicos que nelas podem se alojar e serem levados ao campo, provocando redução na germinação e vigor, e originando focos primários de infecção de doenças. O presente trabalho teve por objetivo desenvolver nova metodologia de diagnóstico com o uso das técnicas moleculares que permitissem detectar e identificar a presença de Psg, Xag e Cff em sementes de soja por meio do desenvolvimento de primers espécie específicos. Os primers desenhados a partir de sequências da região espaçadora 16S-23S RNAr, mostraram-se altamente específicos e sensíveis. O par de primers Curto2f/p322anti gerou um fragmento de 675 pb e capacidade de detecção a partir de 0,01 ng de DNA genômico e aproximadamente 5x103 UFC/PCR; o par de primers Psgl/p322anti gerou um fragmento de 500 pb e o grau mínimo de sensibilidade foi de 1 pg de DNA genômico e cerca de 80 UFC/PCR; o par de primers Xanth2f/p322anti gerou um fragmento de 545 pb e capacidade de detecção a partir de 1 ng de DNA genômico e cerca de 700 UFC/PCR. Posteriormente, as sementes de soja foram infectadas artificialmente nas condições de 1; 0,5 e 0,1% de infecção. Nas amplificações com os primers espécie-específicos desenvolvidos, foi possível detectar as fitobactérias em todos os níveis de infecção testados diretamente do extrato bruto, e nas amplificações após o enriquecimento do extrato (BIO-PCR) o sinal positivo foi potencializado / Abstract: Soybean is considered one of the most important crops in Brazil, due to its high socio-economic value, determined by its several products and sub products and its significant expression on the internal and external market. However, this oleaginous plant is often affected by the occurrence of different diseases, which cause depreciation of the product, reduction in yield and substantial economic losses. Among the main bacterial diseases, the bacterial pustule, caused by Xanthomonas axonopodis pv. glycines (Xag), the bacterial blight caused by Pseudomonas savastanoi pv. glycines (Psg), and bacterial tan spot caused by Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), producing yield losses of up to 40%. The seed health is extremely important since they are considered vehicles of pathogenic agents which can be led to the field, causing germination reduction and vigor; and yielding primary infection of the diseases. This study aimed to develop a new method of diagnosis using molecular tools to detect and identify Psg, Xag or Cff in soybean seeds, through species-specific primers. The primers were designed from sequences of the 16S-23S rRNA and they were highly specific and sensitive. The pair of primers Curto2f/p322anti generated a fragment of 675 bp and was able of detecting down to 0.01 ng of genomic DNA and about 5x103 CFU/PCR; the primer set Psgl/p322anti produced a fragment of 500 bp and reached a detection limit of 1 pg of genomic DNA and about 80 CFU/PCR; and Xanth2f/p322anti yielded a fragment of 545 bp and could detect up to 1 ng of genomic DNA and about 700 CFU/PCR. Subsequently, soybean seeds were artificially infected in the following conditions: 1, 0,5% and 0,1% infection. In the amplifications using the species-specific primers, it was possible to detect the three different phytobacteria at all tested levels of infection directly of the crude extract and in the amplifications after enrichment of the extract (BIO-PCR), the positive signal was enhanced / Mestrado / Genetica de Microorganismos / Mestra em Genética e Biologia Molecular

No description available.

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.

La mancha bacteriana causada por el patógeno Pseudomonas syringae pv. tomato (P. s. tomato) es una de las enfermedades más devastadoras del cultivo del tomate. En este trabajo se demuestra que la sola inhibición de la expresión del gen Ep5C, que codifica una peroxidasa catiónica extracelular, es suficiente para conferir una marcada resistencia a P.s. tomato. Esta inhibición encontrada en las plantas de tomate produce una resistencia que no requiere la activación de las rutas de defensa descritas hasta ahora, controladas por el ácido salicílico y el ácido jasmónico. Así, la inhibición de este gen constituye una nueva herramienta genética para obtener plantas transgénicas resistentes a esta enfermedad. La temprana inducción del gen Ep5C está mediada por el H2O2, una especie reactiva de oxígeno generada durante el curso de u interacciones planta-patógeno. Los mecanismos que controlan la resistencia de las plantas a patógenos necrotrofos constituye uno de los aspectos menos estudiados en la actualidad. La búsqueda de nuevos componentes genéticos que participan en la cascada de señalización de las plantas frente a patógenos constituye uno de los retos de la biología molecular moderna. En este trabajo llevamos a cabo un escrutinio, utilizando plantas transgénicas de Arabidopsis thaliana portadoras del gen de la B-glucoronidasa (GUS) como gen marcador bajo el control del promotor del gen Ep5C, en busca de mutantes alterados en la expresión de dicho gen. En el presente trabajo presentamos la identificación y caracterización de uno de los mutantes, en concreto el mutante ocp3 (overexpressor of cationic peroxidase 3), el cual presenta expresión constitutiva del gen GUS. Las plantas ocp3 muestran una elevada acumulación de H2O2, y se caracterizan por presentar expresión constitutiva de GST1 y PDF1.2, dos genes marcadores de la respuesta defensiva, pero sin embargo no muestra expresión de PR-1, un gen marcador dependiente de la ruta del ácido salicílico (SA). La característic / Coego González, A. (2006). Análisis funcional del gen Ep5C y su implicación en los mecanismos de defensa en plantas [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/1972 / Palancia

Los fenilpropanoides constituyen un grupo de metabolitos secundarios producidos y utilizados por las plantas como parte de la respuesta defensiva tanto constitutiva como inducible. Un gran número de ellos están implicados en la resistencia frente a la enfermedad a diferentes niveles: señalización (ácido salicílico), agentes antimicrobianos (fitoalexinas), y endurecimiento de la pared celular (lignina). Las amidas derivadas del ácido hidroxicinámico (HCAAs) son un conjunto de metabolitos, pertenecientes al grupo de los fenilpropanoides, que desempeñan un importante papel en la defensa de las plantas frente a patógenos y predadores. Las HCAAs se forman a partir de la condensación de tioésteres de hidroxicinamoil-CoA con feniletilaminas, tales como la tiramina. El último paso en la biosíntesis de las HCAAs está catalizado por el enzima tiramina hidroxicinamoil transferasa (THT). En la presente tesis se muestra la identificación y el estudio de cuatro HCAAs, p-cumaroildopamina, feruloildopamina, p-cumaroiltiramina y feruloiltiramina, asociadas a la infección de tomate con la bacteria Pseudomonas syringae pv. tomato. Su identificación y caracterización estructural se han llevado a cabo mediante técnicas de cromatografía líquida de alta resolución y espectrometría de masas (HPLC-MS). Se ha analizado la posible implicación del ácido salicílico y del etileno en la inducción patogénica de dichas moléculas y del enzima responsable de su biosíntesis (THT). Además, se ha estudiado la actividad antioxidante y antibacteriana in vitro de las cuatro HCAAs identificadas. Por último, se han obtenido líneas transgénicas de Arabidopsis thaliana y de tomate que sobreexpresan el gen de la THT, y se han analizado los perfiles cromatográficos de dichas líneas. / Zacarés Sanmartín, L. (2008). Nuevas aportaciones al metabolismo secundario del tomate. Identificación y estudio de moléculas implicadas en la respuesta a la infección con pseudomonas syrinagae pv. tomato [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/3021 / Palancia