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Cheng, Yu Ti. "Dissecting plant innate immunity using SNC1 : a sensitive immune receptor." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/44310.
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Steele, John. "Molecular recognition in plant immunity." Thesis, University of East Anglia, 2016. https://ueaeprints.uea.ac.uk/58564/.
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Plant pathogens constitute a major threat to global food security. The use of naturally resistant crop varieties can limit crop losses, however new races of pathogen can arise that are able to overcome these defences. Plant breeding for race-specific resistance typically relies on disease-resistance genes, which generally encode proteins with nucleotide-binding and leucine-rich repeat domains (NB-LRRs). NB-LRRs are a large of proteins found in both plants and animals, with plant NB-LRRs further classified by the presence of N-terminal coiled-coil or toll-interleukin receptor domains. Although qualitative models exist to describe R-protein regulation and activation, these are predominantly based on genetic and molecular studies. Biochemical investigations into R-protein function have been hampered by difficulties obtaining sufficient yields of material. When suitable material has been identified, biochemical studies have been used to complement well-established in planta assays to validate numerous hypotheses. This work describes the screening processes undertaken to obtain R-protein domains suitable for downstream experiments. Using E. coli for high-throughput screening of constructs from a single R-protein, traditional construct design to investigate multiple R-protein domains and expanding our expression hosts to eukaryotic systems we successfully purified four coiled-coil domains and a single NBARC domain for use in downstream experiments. Characterisation of this NBARC domain by circular dichroism and small-angle X-ray scattering indicates that the protein is well-folded and stable in solution, allowing in vitro investigations. In testing models for R-protein regulation we were able to confirm previous findings, such as low levels of ATPase activity, however we were unable to find evidence for a commonly cited method of signal repression. A preliminary crystal structure of the NBARC domain shows significant similarity to Apaf-1, and highlights the importance of conserved motifs in NBARC architecture. The tools presented here should prove a valuable resource to complement existing models to better understand the structure, biochemistry, and ultimately regulation of plant R-proteins.
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Gao, Qing-Ming. "GLYCEROLIPIDS AND THE PLANT CUTICLE CONTRIBUTE TO PLANT IMMUNITY." UKnowledge, 2012. http://uknowledge.uky.edu/plantpath_etds/4.
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The conserved metabolites, oleic acid (18:1), a major monounsaturated fatty acid (FA), and glycerol-3-phosphate (G3P) are obligatory precursors of glycerolipid biosynthesis in plants. In Arabidopsis, the SSI2-encoded SACPD is the major isoform that contributes to 18:1 biosynthesis. Signaling induced upon reduction in oleic acid (18:1) levels not only upregulates salicylic acid (SA)-mediated responses but also inhibits jasmonic acid (JA)- inducible defenses. I examined the transcription profile of ssi2 plants and identified two transcription factors, WRKY50 and WRKY51. Although the ssi2 wrky50 and ssi2 wrky51 plants were constitutively upregulated in SA-derived signaling, they were restored in JAdependent defense signaling. Not only did these plants show JA-inducible PDF1.2 expression, but they were also restored for basal resistance to the necrotrophic pathogen, Botrytis cinerea. Overall, my results show that the WRKY50 and WRKY51 proteins mediate both SA- and low 18:1-dependent repression of JA signaling in Arabidopsis plants.
My studies also show that cellular G3P levels are important for plant defense to necrotrophic pathogens. I showed that G3P levels are induced in Arabidopsis in response to the necrotrophic fungal pathogen B. cinerea. G3P-dependant induction of basal defense is not via the activities of other defense-related hormones such as SA, JA or the phytoalexin camalexin. Arabidopsis mutants unable to accumulate G3P (gly1, gli1) showed enhanced susceptibility to B. cinerea.
Previous studies in our lab identified acyl-carrier protein 4 (ACP4), a component of FA and lipid biosynthesis, as an important regulator of plant systemic immunity. ACP4 mutant plants were defective in systemic acquired resistance (SAR) because they contained a defective cuticle. I further investigated the role of the plant cuticle in SAR by studying the involvement of long-chain acyl-CoA synthetases (LACS), a gene family involved in long-chain FA and cuticle biosynthesis, in SAR. In all, eight lacs mutants (lacs1, lacs2, lacs3, lacs4, lacs6, lacs7, lacs8, lacs9) were isolated and characterized. Six mutants were compromised in SAR. Together, my studies show that the various LACS isoforms contribute differentially to both cuticle formation and systemic immunity in Arabidopsis.
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Scandolera, Tiffanie. "Interactions plante-virus : impacts d'un fort taux de CO2 atmosphérique et de fortes températures sur la résistance/sensibilité des plantes aux virus dans le contexte du changement climatique." Electronic Thesis or Diss., université Paris-Saclay, 2025. http://www.theses.fr/2025UPASB014.
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Comprendre et anticiper l’impact du changement climatique sur les interactions plantes-bioagresseurs est un enjeu majeur pour l’agriculture de demain. Les modèles de prédiction climatiques prévoient une hausse de la concentration en CO₂ atmosphérique qui pourrait passer de 400 µl.L-1 en 2014, à 1000 µL.L-1 en 2100 ainsi qu’une élévation de la température moyenne annuelle de 4.6°C d’ici 2100. Les plantes sont directement impactées par la hausse du taux de CO₂ atmosphérique et de la température.L’objectif de ce projet de thèse est d’étudier l’impact d’un fort CO₂ et de températures élevées sur le niveau de résistance/sensibilité aux virus des plantes. Le modèle d’étude est le haricot commun (Phaseolus vulgaris L.), une plante d’intérêt agronomique, en réponse à l’infection par un comovirus, le bean pod mottle virus (BPMV, Comovirus siliquae). Deux génotypes de haricot commun ont été étudiés dans le cadre de ce projet: un génotype naturellement résistant au BPMV (BAT93), et un génotype naturellement sensible (Black Valentine).Cette étude est décomposée en trois parties: dans un premier temps, une approche par sélection a priori de gènes impliqués dans différentes voies de défenses aux virus a été choisie pour étudier 1/ l’impact d’un fort CO₂ et 2/ l’impact de températures élevées sous forme de vagues de chaleur sur la résistance/sensibilité au BPMV chez les deux génotypes de P. vulgaris Dans un deuxième temps, une étude plus exhaustive par RNAseq a été réalisée afin d’étudier 3/ l’effet combiné de fort CO₂ et vague de chaleur sur la résistance au BPMV chez le génotype naturellement résistant de P. vulgaris, BAT93Understanding and anticipating the impact of climate change on plant-pathogen interactions is a major challenge for the agriculture of tomorrow. Climate predictive models forecast an increase in atmospheric CO₂ concentration from 400 µL.L-1 in 2014 to 1000 µL.L-1 by 2100, as well as a rise in the average annual temperature of 4.6°C by 2100. Plants are directly affected by the increase in CO₂ levels and temperature. The objective of this doctoral project is to study the impact of high CO₂ and elevated temperatures on the level of plant resistance/susceptibility to viruses. The model organism is the common bean (Phaseolus vulgaris L.), an agronomically significant plant, in response to infection by a comovirus, the bean pod mottle virus (BPMV, Comovirus siliquae).Two common bean genotypes were studied in this project: one that is naturally resistant to BPMV (BAT93) and one that is naturally susceptible (Black Valentine).This study is divided into three parts. First, an approach based on the a priori selection of genes involved in different defense pathways against viruses was chosen to study 1/ the impact of high CO₂ and 2/ the impact of heat waves on resistance/susceptibility to BPMV in in both genotypes of P. vulgaris.Second, a more comprehensive approach was performed by RNAseq, in order to study 3/ the combined effect of high CO₂ and heat waves on resistance to BPMV in the naturally resistant genotype of P. vulgaris, BAT93
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Prince, David. "Dissecting the role of plant immunity in plant-aphid interactions." Thesis, University of East Anglia, 2012. https://ueaeprints.uea.ac.uk/42420/.
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Aphids are economically important phloem-feeding insects that cause loss in plant productivity worldwide. This occurs through the removal of photoassimilates and the vectoring of hundreds of plant viruses. Plants possess a complex immune system in order to defend themselves from a range of pathogens including bacteria and fungi. I aimed to discover if this immune system was also involved in defence against aphids. I found that aphids have proteins that trigger plant immune responses. The aphid Myzus persicae contains several protein elicitors with varying molecular weights. These proteins are perceived by the plants Nicotiana benthamiana and Arabidopsis thaliana. In A. thaliana the perception of a 3 to 10 kDa elicitor fraction requires the leucine-rich repeat receptor-like kinase (LRR-RLK) BAK1, as a mutant in this gene was deficient in immune responses activated by this elicitor. Plant recognition of the elicitor is unlikely to depend on a single non-arginine-asparate (non-RD) RLK. In addition, aphids possess the means to modulate the plant immune response. I helped to identify three aphid effectors that modulate plant processes. I then investigated the role of one of these effectors, a M. persicae chemosensory protein (CSP) known as Mp10, in suppressing the immune responses triggered by the aphid elicitors. Mp10 is likely to disrupt the function of plant genes near the top of the immune signalling cascade in N. benthamiana in order to suppress elicitor-triggered immunity. Surprisingly, the homologs of this CSP in other aphids also show the same ability to suppress plant immune responses, suggesting an important role for Mp10 in plant-aphid interactions. This is the first report of a role for elicitor recognition by plants in aphid defence, the use of plant cell surface receptors to detect insects, and aphids’ attempts to suppress plant immunity.
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Goritschnig, Sandra. "Protein modification in plant innate immunity." Thesis, University of British Columbia, 2006. http://hdl.handle.net/2429/30887.
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Plant diseases cause major crop losses worldwide. Crop protection
strategies enhancing the plants' own defence mechanisms could be a sustainable
solution to ensure future food supply. This thesis describes my research effort to
better understand the innate defence mechanisms in plants.
Specific resistance responses towards invading pathogens are mediated by
Resistance (R) proteins. They recognize pathogen-derived molecules and activate
signalling cascades, initiating physiological responses to limit pathogen spread in
infected cells while minimizing harmful effects on the rest of the plant.
We use the unique gain-of-function R gene snd as a tool to identify
components of resistance signalling in Arabidopsis thaliana. In a screen for
suppressors of snc7-mediated constitutive resistance, we identified a number of
modifier of snd (mos) mutants. My thesis focuses on the identification and
characterization of mos5 and mos8. Both mutations partially suppress sndassociated
morphological phenotypes and revert susceptibility to virulent
pathogens to wild type levels.
mos5 contains a deletion in one of two ubiquitin activating enzyme genes in
Arabidopsis. The mutation in mos5 lies in a putative binding domain, potentially
disrupting interaction with downstream ubiquitin acceptors. The mos5 single
mutant displays enhanced susceptibility to virulent bacteria, as well as to bacteria
carrying the effector protease AvrRpt2, indicating a role of ubiquitination in both
specific and basal resistance. A mutation in the mos5 homolog UBA2 does not
affect resistance, however, a double mutant mos5 uba2 is lethal, indicating that the
two genes are partially redundant.
mos8 is allelic to enhanced response to abscisic acid 1 (eral), which
encodes the beta subunit of protein farnesyltransferase. Mutations in the gene are
known to affect development and abscisic acid signalling. mos8 displays enhanced
susceptibility to virulent and avirulent pathogens and acts additively with NPR1.
Defects in geranylgeranylation, a protein modification similar to farnesylation, do
not affect resistance responses against virulent or avirulent pathogens. Taken together, my data reveals the importance of post-translational
modification of yet to be identified regulatory proteins in plant innate immunity.
Further research will aim at unravelling the mechanisms by which mos5 and mos8
affect resistance signalling.Science, Faculty of
Botany, Department of
Graduate
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Truman, William Matthew Donald. "Signalling pathways underylying plant innate immunity." Thesis, Imperial College London, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429264.
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Escouboué, Maxime. "La phosphorylation de l'effecteur PopP2 de Ralstonia solanacearum par des MAPKs immunitaires potentialise ses fonctions de virulence et limite sa reconnaissance chez Arabidopsis." Thesis, Toulouse 3, 2019. http://www.theses.fr/2019TOU30041.
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Les bactéries phytopathogènes injectent des facteurs de virulence (appelés effecteurs) dans leurs cellules hôtes pour interférer avec de nombreuses voies de signalisation immunitaires. La bactérie Gram négative Ralstonia solanacearum est l'agent causal du flétrissement bactérien. Parmi ses nombreux effecteurs de type III figure PopP2 dont les fonctions de virulence et d'avirulence ont été largement étudiées précédemment. PopP2 cible les facteurs de transcription de défense WRKY et les acétyles pour inhiber la résistance basale (PTI). Au cours de ce travail, nous montrons que la phosphorylation de PopP2 in planta au niveau de trois motifs SP nécessite la présence d'un domaine de type " MAPK-docking domain " situé dans son extrémité N-terminale. Bien que la phosphorylation de PopP2 n'affecte pas sa reconnaissance chez Arabidopsis par le complexe d'immuno-récepteurs RPS4/RRS1-R, ses fonctions de virulence dépendent strictement de cette modification. De façon surprenante, les phosphomutants PopP2, incapables d'être phosphorylés in planta, ont un comportement de protéine avirulente chez un grand nombre d'accessions, indépendamment de la paire RPS4/RRS1-R. Par le biais de différentes approches biochimiques et de microscopie confocale, nous montrons que PopP2 est un substrat de différentes MAPKs associées à la réponse immunitaire et notamment d'AtMPK3 avec qui l'effecteur interagit physiquement au sein du noyau. De façon intéressante, l'activation des activités des MAPKs au cours de la PTI coïncide avec la stimulation du niveau de phosphorylation du PopP2. Une analyse RNA-seq indique que la phosphorylation de PopP2 participe à la dérégulation de nombreux gènes liés aux réponses de défense. Des hypothèses concernant la façon dont le niveau de phosphorylation de PopP2 modulerait ses activités in planta sont présentées. Globalement, ce travail révèle une stratégie de virulence utilisée par un effecteur bactérien qui exploite les MAPKs associées à l'immunité pour (i) potentialiser ses fonctions de virulence et (ii) limiter sa détection chez l'hôteMicrobial pathogens infect host cells by delivering virulence factors (effectors) that interfere with defense. The Gram-negative Ralstonia solanacearum is the causal agent of bacterial wilt. The well-characterized PopP2 effector form R. solanacearum binds to and acetylate WRKY defensive transcription factors to dampen basal defense responses. In this work, we show that PopP2 phosphorylation on three SP motifs involves a MAPK-docking like domain located in its N-terminus. Although PopP2 phosphorylation does not affect its avirulence activity in Arabidopsis expressing the RPS4/RRS1-R immune receptor complex, its virulence functions strictly depend on this modification. Through different biochemical and confocal microscopy approaches, we show that PopP2 is a substrate of different MAPKs associated with the immune response and in particular, AtMPK3 with which the effector physically interacts in the plant nucleus. Interestingly, activation of MAPK activities during establishment of PTI coincides with the stimulation of the phosphorylation level of PopP2. An RNA-seq analysis indicates that the phosphorylation of PopP2 contributes to the deregulation of many genes related to defense responses. Hypotheses about how PopP2's phosphorylation level would modulate its activities in planta are presented. Overall, this work reveals a virulence strategy used by a bacterial effector that exploits MAPKs associated with immunity to (i) potentiate its virulence functions and (ii) limit its detection in the host
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Case, Olivia Hildegard. "An assessment of medicinal hemp plant extracts as natural antibiotic and immune modulation phytotherapies." Thesis, University of the Western Cape, 2005. http://etd.uwc.ac.za/index.php?module=etd&.
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This study aimed to evaluate the antimicrobial efficacy of medicinal hemp plant extracts to determine the antibacterial effects of indigenous Sansevieria species and exotic Cannabis sativa phytotherapy varieties. This study also assessed whether aqueous o
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Camargo, Ramírez Rosany del Carmen. "Function of microRNAs in plant innate immunity." Doctoral thesis, Universitat Autònoma de Barcelona, 2017. http://hdl.handle.net/10803/405716.
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Esta tesis aborda el estudio de miARNs en la inmunidad innata en plantas. El trabajo se ha desarrollado en arroz (Capítulo I y Capítulo II) y en Arabidopsis (Capítulo III), En el capítulo I se describe la identificación y caracterización funcional de nuevos miARNs de arroz en su interacción con el hongo Magnaporthe oryzae. Este hongo es responsable de la piriculariosis, una de las enfermedades más devastadoras para el cultivo del arroz a nivel mundial. A partir de la información generada mediante secuenciación masiva de bibliotecas de pequeños ARNs de arroz, se seleccionaron secuencias candidatas a representar nuevos miARNs de arroz, habiéndose estudiado 5 de estos candidatos (miR-64, miR-75, miR-96, miR-98 y miR-203). La obtención de líneas transgénicas de arroz ha permitido demostrar que la sobreexpresión de MIR-64 y MIR-75 confiere resistencia a M. oryzae, tratándose por lo tanto de miARNs que funcionan como reguladores positivos en la respuesta inmune de arroz. Por otra parte, la sobreexpresión de MIR-96, MIR-98 o MIR-203 aumenta la susceptibilidad a la infección por M. oryzae en plantas de arroz (reguladores negativos de la respuesta inmune). El análisis de mutantes de arroz afectados en la biogénesis de miARNs (mutantes dcl1, dcl3 y dcl4) indican que la producción del miARN maduro miR-64, miR-75 o miR-96 es dependiente de DCL3 y/o DCL4, lo cual apoya la idea de que se trata de nuevos miARNs de arroz. Además, mediante edición génica por CRISPR/Cas9, se ha comprobado que una delección de 22 nucleótidos en el precursor miR-75 resulta en un fenotipo de susceptibilidad a M. oryzae (Capítulo II), lo que concuerda con el fenotipo de resistencia que se observa en las plantas que sobreexpresan este miARN.
En el capítulo III se ha estudiado la función de miR858 en la inmunidad innata de Arabidopsis thaliana frente a la infección por hongos patógenos. Este miARN reprime la expresión de factores de transcripción de tipo MYB que actúan como activadores de la expresión de genes que participan en la biosíntesis de flavonoides. Cuando la actividad del miR858 se encuentra bloqueada por la expresión de un gen de imitación de díana (plantas MIM858), las plantas son resistentes a la infección por hongos patógenos (Plectosphaerella cucumerina, Fusarium oxysporum f. sp. Conglutinans and Colletotrichum higginsianum), mientras que la sobreexpresión de este miARN confiere mayor susceptibilidad a la infección. Además, la interferencia con la actividad de miR858, y consiguiente aumento de la expresión de genes MYB, en las plantas MIM858 afecta de manera importante el metabolismo de fenilpropanoides, priorizándose la síntesis y acumulación de flavonoides, a expensas de la síntesis de precursores de lignina. La actividad antifúngica que se observa para kaempferol, naringenina (flavonoides) y ácido p-cumárico, explicaría el fenotipo de resistencia a la infección por hongos que se observa en las plantas MIM858.
En su conjunto, los resultados obtenidos en este trabajo demuestran que los miARNs son componentes importantes en la resistencia/susceptibilidad a la infección por patógenos fúngicos en plantas de arroz y Arabidopsis. Un mayor conocimiento de función de miARNs en la inmunidad innata de las plantas, y de los procesos que son regulados por estos riboreguladores, puede ser de utilidad en el diseño de nuevas estrategias para el control de enfermedades en plantas.This thesis comprises the study of miRNAs in innate immunity in plants. The work has been developed in rice (Chapter I and Chapter II) and in Arabidopsis (Chapter III), model systems used in studies of functional genomics in monocotyledonous and dicotyledonous species, respectively. Chapter I describes the functional identification and characterization of new rice miRNAs in their interaction with the fungus Magnaporthe oryzae. This fungus is responsible for blast disease, one of the most devastating diseases for rice cultivation worldwide. From the information generated by high-throughput sequencing of small rice RNA libraries, candidate sequences to represent novel rice miRNAs were selected. In this work 5 of these candidates have been studied (miR-64, miR-75, miR-96, miR-98 and miR-203). Obtaining transgenic rice lines has demonstrated that the overexpression of MIR-64 and MIR-75 confers resistance to M. oryzae, therefore these miRNAs function as positive regulators in the rice immune response. Moreover, overexpression of MIR-96, MIR-98 or MIR-203 increase susceptibility to M. oryzae in rice plants (negative regulators of immune response). Analysis of rice mutants affected in the miRNA biogenesis (dcl1, dcl3 and dcl4 mutants) indicate that the mature miRNA production of miR-64, miR-75 or miR-96 depends on DCL3 and/or DCL4, which supports the idea that they are novel rice miRNAs. Furthermore, by gene editing using CRISPR/Cas9, it has been found that a 22 nucleotides deletion in miR-75 precursor results in a susceptibility phenotype under M. oryzae infection (Chapter II), in agreement with a resistance phenotype that was observed in overexpressor plants for this miRNA. In chapter III, the miR858 function in Arabidopsis thaliana innate immunity to infection by pathogenic fungi was studied. This miRNA represses the expression of MYB transcription factors, which act as activators of the expression of genes involved in flavonoids biosynthesis. Plants are resistant to infection by pathogenic fungi (Plectosphaerella cucumerina, Fusarium oxysporum f. sp. Conglutinans and Colletotrichum higginsianum) when the activity of miR858 is blocked by the expression of target mimicry (MIM858 plants), while the overexpression of this miRNA confers greater susceptibility to infection. Additionally, interference with miR858 activity and consequent increase of MYB gene expression in MIM858 plants significantly affects phenylpropanoids metabolism, favoring the synthesis and accumulation of flavonoids, and disfavoring the synthesis of lignin precursors. The antifungal activity that was observed for Kaempferol, naringenin (flavonoids) and p-Coumaric acid, would explain the resistant phenotype by fungi infection which is observed in the MIM858 plants. Altogether, the results obtained in this work demonstrate that miRNAs are an important component in the resistance/susceptibility to infection by pathogenic fungi in Arabidopsis and rice plants. Greater knowledge of miRNA function in plant innate immunity and processes that are regulate by these riboregulators, can be useful in the design of new strategies for the control of diseases in plants.
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Baldrich, Patricia. "Role of microRNAs in plant innate immunity." Doctoral thesis, Universitat Autònoma de Barcelona, 2015. http://hdl.handle.net/10803/315463.
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Los pequeños ARNs son ARNs cortos no codificantes que regulan la expresión génica en la mayoría de eucariotas. Las plantas tienen dos clases de pequeños ARNs, los microARNs (miRNAs) y los pequeños ARNs de interferencia (siRNAs), que se diferencian por su biogénesis y su modo de acción.
Actualmente se estima que las pérdidas debidas a patógenos y plagas alcanzan el 50-80%, siendo uno de los factores limitantes de la producción y causando graves pérdidas económicas. Tenemos pues la necesidad de mejorar el conocimiento de los mecanismos de defensa y desarrollar nuevas estrategias para la protección de los cultivos. Con el fin de ampliar los conocimiento en este sector, se han llevado a cabo estudios con Arabidopsis y arroz, los dos sistemas modelo usados en genómica funcional en plantas dicotiledóneas y monocotiledóneas.
En el primer capítulo, se analizaron las alteraciones en la acumulación de pequeños ARNs, incluyendo miRNAs, en respuesta al tratamiento con elicitores en plantas de Arabidopsis. Entre los miRNAs regulados por elicitores se encuentra miR168, que regula ARGONAUTE1, el componente principal del complejo RISC (RNA-induced silencing complex) de la maquinaria de funcionamiento de miRNAs. Los microarrays permitieron, además de analizar miRNAs conocidos, la identificación de un pequeño ARN incorrectamente anotado como miRNA en el registro miRBase. Se demostró que este pequeño ARN es un pequeño ARN heterocromático (hc-siRNA) denominado siRNA415.
En el segundo capítulo, se usaron librerías de secuenciación masiva de pequeños ARNs para la identificación global de miRNAs de arroz regulados por elicitores fúngicos. Esto permitió también describir 9 miRNAs no caracterizados previamente. Combinando dichas librerías con el análisis de nuestros degradomas, se identificaron sus respectivos targets y se observó la existencia de redes reguladoras enriquecidas en miRNAs regulados por elicitores. Específicamente, se identificó un número importante de miRNAs y sus genes diana, implicados en las rutas de biosíntesis de pequeños ARNs, incluyendo miRNAs, hc-siRNAs y “trans-acting siRNAs” (ta-siRNAs). Así mismo se presentan evidencias de miRNAs y sus genes diana implicados en la señalización mediada por hormonas así como en la interacción entre rutas hormonales, demostrando así un gran potencial en la regulación de la inmunidad del arroz. Asimismo se describe la regulación de genes de arroz que contienen “Conserved-Peptide upstream Open Reading Frame” (CPuORF) mediada por miRNAs. Esto sugiere la existencia de una red reguladora nueva que integra la función de miRNAs y CPuORF en plantas. El conocimiento adquirido en este estudio ayudará al la comprensión de los mecanismos de defensa mediados por miRNAs, así como a desarrollar estrategias apropiadas a la protección del arroz.
En el tercer capítulo, se uso la combinación de herramientas bioinformáticas y experimentales para el análisis de miRNAs policistrónicos en arroz, observando la existencia de 23 loci con la habilidad de formar la típica estructura de horquilla de los precursores de miRNAs. Se presentan evidencias experimentales de 7 miRNAs policistrónicos que contienen tanto miRNAs homólogos como miRNAs no homólogos. Se demostró también un patrón de conservación en diferentes especies de arroz (Oryza sativa) cuyo genoma es AA, no presente en especies primitivas de arroz.
Conjuntamente, los resultados obtenidos en este trabajo apoyan la idea que los miRNAs pueden ser considerados como componentes de la respuesta de las plantas a la infección por patógenos, posiblemente actuando como nodos de regulación de diferentes procesos fisiológicos durante la adaptación de las plantas a la infección.Small RNAs (sRNAs) are short non-coding RNAs that guide gene silencing in most eukaryotes. Plants have two main classes of sRNAs, microRNAs (miRNAs) and small interfering RNAs (siRNAs), which are distinguished by their mode of biogenesis and mechanisms of action. In this day and age, crop losses due to pathogens and pests are estimated from 50% to 80%, factors limiting crop production and causing important economical losses. There is then an imperative need to improve our knowledge in defense mechanisms and to develop novel strategies for crop protection. To improve the understanding in this field, we carried out studies in Arabidopsis and rice plants, the two model systems used for functional genomic studies in dicot and monocot plant species. In the first chapter, we analyzed alterations on the accumulation of smRNAs in response to elicitor treatment, including miRNAs, in Arabidopsis plants. Among the elicitor-regulated miRNAs was miR168 which regulates ARGONAUTE1, the core component of the RNA-induced silencing complex involved in miRNA functioning. In addition to known miRNAs, microarray analysis allowed the identification of an elicitor-inducible small RNA that was incorrectly annotated as a miRNA in the miRBase registry. We demonstrated that this smRNA, is a heterochromatic-siRNA (hc-siRNA) named as siRNA415. In the second chapter, we used deep sequencing of small RNA libraries for global identification of rice miRNAs that are regulated by fungal elicitors. We also describe 9 previously uncharacterized miRNAs. Combined small RNA and degradome analyses revealed regulatory networks enriched in elicitor-regulated miRNAs supported by the identification of their corresponding target genes. Specifically, we identified an important number of miRNA/target gene pairs involved in small RNA pathways, including miRNA, heterochromatic and trans-acting siRNA pathways. We present evidence for miRNA/target gene pairs implicated in hormone signaling and cross-talk among hormone pathways having great potential in regulating rice immunity. Furthermore, we describe miRNA-mediated regulation of Conserved-Peptide upstream Open Reading Frame (CPuORF)-containing genes in rice, which suggests the existence of a novel regulatory network that integrates miRNA and CPuORF functions in plants. The knowledge gained in this study will help in understanding the underlying regulatory mechanisms of miRNAs in rice immunity and develop appropriate strategies for rice protection. In the third chapter, we used a combination of bioinformatic tools and experimental analyses for the discovery of new polycistronic miRNAs in rice, revealing 23 loci with the ability to form the typical hairpin structure of miRNA precursors in which two or more mature miRNAs mapped along the same structure. Evidence is presented on the polycistronic nature of 7 miRNA precursors containing homologous or non-homologous miRNA species. We also demonstrated a pattern of conservation in the genome of rice (Oryza sativa) species that have an AA genome, but not in primitive rice species. Collectivelly, results obtained in this work support the notion that miRNAs might be considered as components of the plant response to pathogen infection, possible acting as regulatory nodes of different physiological processes during plant adaptation to infection conditions.
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Kancy, Stephanie Jayne Thorley. "The role of histone acetyltransferases in plant immunity." Thesis, University of Warwick, 2017. http://wrap.warwick.ac.uk/102601/.
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Activation of plant defence responses requires significant transcriptional reprogramming to mount an effective response to pathogens. This response must be finely balanced with growth and development processes to ensure optimal allocation of cellular resources. A fundamental mechanism of gene expression regulation is covalent modification of histones. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) antagonistically control the acetylation levels of histones at specific genomic loci to ultimately affect gene expression. This thesis focuses on histone acetylation as a mechanism by which plants mount an effective immune response. In Chapter 3, a reverse genetic screen of Arabidopsis HAT mutants is presented where a negative regulator (HAM2) of defence against the plant pathogen Pto DC3000 was identified. Whilst mutants of the negative regulator (ham2 ) demonstrate enhanced resistance to P. syringae, their susceptibility to the necrotrophic pathogen B. cinerea is unchanged. Alongside the immunity phenotype, ham2 plants exhibit increased adult leaf surface area, fresh weight and root length. Since ham2 is the only known Arabidopsis mutant with increased immunity and growth, it represents a promising target in an agricultural context. In Chapter 4, homology models of A. thaliana, B. napus and S. lycopersicum HAM2 proteins were created, supported by a series of cheminformatics and in silico docking methods, to identify chemical inhibitors for future agricultural applications. Finally, the role of Arabidopsis HATs in effector-triggered immunity was investigated in Chapter 5. Here, HAG1 was identified as a key positive regulator of effector-triggered responses. Overall, this thesis contributes to our understanding of the role of HAM2 and HAG1 histone acetyltransferases in plant immunity, and presents HAM2 as a novel target in an agricultural context.
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Huang, Shuai. "Using chemical genetics to discover regulators in plant immunity." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/44065.
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Kneeshaw, Sophie. "Molecular mechanisms of redoxin-mediated signalling in plant immunity." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/18754.
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Posttranslational modification (PTM) of proteins is essential to creating a diverse proteome with the complex functions necessary to regulate key cellular processes. Redox-based PTMs exhibit many desirable characteristics to finely modulate transcriptional regulators; they occur rapidly and can alter protein conformation, localisation and activity. The plant immune system offers an excellent model in which to study redox-based modifications due to the rapid accumulation of oxidising agents that occurs during immune invasion. This so-called “oxidative burst” causes spontaneous oxidation of cysteine residues that are present in many regulatory proteins. These modifications fine-tune the activities of proteins that harbour them, enabling them to act in a concerted effort to reprogram the transcriptome, prioritising the expression of immune-related genes over housekeeping genes. Disulphide bonds (S-S) and S-nitrosothiols (SNO, i.e. the addition of an NO group to a cysteine moiety) have been shown to play particularly important roles in plant immunity. However, what still remains unclear is how these redox-based PTMs are rendered reversible, enabling them to act as molecular signalling switches. The work presented in this thesis explores a class of enzymes that are responsible for controlling the cellular levels of protein oxidation: the Thioredoxins. In addition to their well-established role in reducing disulphide bonds, I demonstrate in Chapter 3 that Thioredoxins are able to reverse protein S-nitrosylation during plant immune signalling. Immune-inducible Thioredoxin-h5 (TRXh5) was shown to be unable to restore immunity in gsnor1 mutants that display excessive accumulation of the NO donor S-nitrosoglutathione, but rescued impaired immunity and defence gene expression in nox1-mutants that exhibit elevated levels of free NO. This data indicates that TRXh5 discriminates between protein-SNO substrates to provide previously unrecognized specificity and reversibility to protein-SNO signalling in plant immunity. Furthermore, data is presented to show that TRXh5 reversed the effects of S.nitrosylation on many immune-related transcriptional regulators in vitro, forming the initial stages of an investigation into which proteins and pathways might be controlled by reversible S-nitrosylation in plant immunity (Chapters 3 & 4). Although the majority of transcriptional regulators are likely modified at their site of action, the nucleus, very little is currently known about nuclear redox signalling in plants. Therefore, in Chapter 5 a subclass of theThioredoxin superfamily was studied, the Nucleoredoxins, which have previously been shown to display disulphide reduction activity and localise in part to the nucleus. Here it is revealed that the activity and nuclear accumulation of Nucleoredoxin 1 (NRX1) is induced by the plant leaf pathogen Pseudomonas syringae, suggesting a key role for this protein in immune signalling. Target-capture experiments and subsequent mass spectrometry analysis identified the first in vitro targets of NRX1 and revealed many proteins with roles in oxidative stress, including the hydrogen peroxide scavenger Catalase 2 (CAT2). Moreover, overexpression of NRX1 was shown to be able to rescue the enhanced cell death phenotype of cat2 knockout mutants in response to the oxidative stressor, methyl viologen. Accordingly, nrx1 knockout mutants also exhibited an enhanced cell death phenotype in response to methyl viologen treatment. Together, these data indicate that NRX1 plays a key role in the control of oxidative stress-mediated cell death, potentially through direct regulation of Catalase proteins. Taken together, the work in this thesis implicates members of the Thioredoxin family as key regulators of transcriptional reprogramming during plant immunity and uncovers a novel role for Thioredoxin superfamily member, NRX1, in the control of oxidative stress.
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Skelly, Michael J. "Interplay between S-nitrosylation and SUMOylation in plant immunity." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/17924.
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Post-translational protein modifications (PTM) vastly increase the complexity and functional diversity of the proteome, to precisely regulate crucial cellular processes. The plant immune system is composed of complex signalling networks that are influenced by various PTMs. Activation of plant immunity is associated with a rapid burst of nitric oxide (NO), which can covalently modify cysteine thiols within target proteins by a process termed S-nitrosylation to form S-nitrosothiols (SNOs), constituting a redox-based PTM. Another key PTM involved in plant immunity is SUMOylation, an essential mechanism involving the conjugation of the small ubiquitin-like modifier (SUMO) peptide to lysine residues within target proteins. Although the targets and mechanisms of S-nitrosylation and SUMOylation are becoming evident, how these key PTMs are themselves regulated remains obscure. Work presented in this thesis reveals that during plant immune signalling, the sole Arabidopsis thaliana SUMO conjugating enzyme, SUMO CONJUGATING ENZYME 1 (SCE1), is S-nitrosylated at a highly conserved, but previously uncharacterized cysteine. S-nitrosylation of SCE1 was shown to inhibit its SUMO conjugating activity in vitro and mutational analysis revealed that the site of this modification, Cys139, is not required for enzyme activity but rather constitutes a redox-sensitive inhibitory switch. Generation and characterization of transgenic Arabidopsis plants overexpressing both wild-type and mutant forms of SCE1 revealed that Cys139 is required for efficient immunity against bacterial pathogens. Furthermore, after immune activation, S-nitrosylation of this residue inhibits global SUMOylation of proteins. These results provide evidence of a novel means of crosstalk between S-nitrosylation and SUMOylation in the context of plant immunity. The abundant cellular antioxidant, glutathione (GSH), is S-nitrosylated to form S-nitrosoglutathione (GSNO), which is thought to constitute a stable reservoir of NO bioactivity. In Arabidopsis, GSNO levels are controlled by the enzyme S-NITROSOGLUTATHIONE REDUCTASE 1 (GSNOR1), which indirectly influences the levels of protein SNOs. In this study, transgenic plants overexpressing FLAG-epitope tagged GSNOR1 were generated in various mutant backgrounds, including nitric oxide overproducer 1 (nox1), to further investigate the roles of GSNOR1 and NO in plant immunity. It was shown that ectopic GSNOR1 expression completely recovers developmental and disease susceptibility phenotypes of gsnor1, but not nox1 mutant plants, highlighting in vivo differences between accumulation of GSNO and free NO. Surprisingly, elevated NO levels in nox1 plants promote S-nitrosylation of GSNOR1, inhibiting its enzymatic activity. This suggests a previously unreported means by which NO might regulate its own bioavailability. Further work in this study revealed that recombinant GSNOR1 can be SUMOylated in vitro, which appeared to increase its enzymatic activity. Several potential SUMO modification sites were identified within GSNOR1 and mutational analysis revealed that at least one of these, Lys191, is SUMOylated. Co-immunoprecipitation experiments revealed that transgenic GSNOR1 might be SUMOylated in vivo, although the site(s) and biological function of SUMOylation were not identified. Nonetheless, these results reveal another possible layer of interplay between S-nitrosylation and SUMOylation.
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Li, Yuan. "Redox regulation of salicylic acid synthesis in plant immunity." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/19578.
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Salicylic acid (SA) is essential to the establishment of both local and systemic acquired resistance (SAR) against a wide range of phytopathogens. Isochorismate synthase 1 (ICS1) is the key enzyme involved in the synthesis of SA and it is transcriptionally activated by the regulatory proteins SAR deficient 1 (SARD1) and Calmodulin binding protein 60g (CBP60g). It has been demonstrated previously that the loss-of-function mutant, S-nitrosogluthione reductase 1-3 (gsnor1-3), increased cellular levels of S-nitrosylation. Significantly, accumulation of both free SA and its storage form SA-glucoside (SAG), were substantially reduced, disabling multiple SA-dependent immune responses. However, the molecular mechanism underlying this observation remains to be established. Our data suggests that the transcription of ICS1 and it regulators, SARD and CBP60g, are reduced in the gsnor1-3 mutant, implying that increased cellular S-nitrosylation blunts the expression of ICS1 by reducing the transcription of its activators. We demonstrated that SARD1 is S-nitrosylated in vitro resulting in inhibition of its DNA binding activity. Further, Cys438 of SARD1 was found to be the site of S-nitrosylation, demonstrated by the observation that the SARD1 C438S mutant was insensitive to NO regulation in regard to DNA binding activity.
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Shirsekar, Gautam Shashikant. "Ubiquitination in Innate Immunity of Rice (Oryza sativa)." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1383664921.
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Fang, Xu. "Genetic and molecular analysis of resistance protein mediated plant immunity." Thesis, University of British Columbia, 2014. http://hdl.handle.net/2429/50783.
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Upon recognition of pathogen effectors, plant resistance proteins trigger strong defence responses that restrict the growth and spread of pathogens. Most of these immune receptors are nucleotide-binding (NB) and leucine-rich repeat (LRR) domain-containing proteins (NLRs). SNC1 (Suppressor of npr1, constitutive 1) is an Arabidopsis Toll/interleukin-1 Receptor (TIR)-type NLR that was originally identified through a gain-of-function autoimmune mutant, snc1, from a forward genetic screen. My Ph.D. thesis is comprised of three projects, all taking advantage of the unique autoimmune phenotypes of snc1 to enable efficient genetic screening.
Firstly, MOS12 was identified from the snc1 suppressor screen, which encodes a protein homologous to cyclin L of mouse and human. MOS12 is required for both SNC1- and RPS4- mediated defense response through its involvement in alternative splicing of SNC1 and RPS4. MOS12 associates with the MAC in planta and MAC components also contribute to proper splicing of SNC1 and RPS4. This study provides the emerging regulatory details of alternative splicing of certain NLR genes.
Secondly, the transcription factor bHLH84 was isolated from a reverse genetic screen by its ability to confer enhanced immunity when overexpressed. bHLH84 and its homologs function redundantly in SNC1- and RPS4-mediated defense response. While bHLH84 does not seem to regulate the expression of NLR-encoding genes directly, it associates with SNC1 and RPS4 to fulfil their function in plant immunity. Being a transcription activator, bHLH84 associates with nuclear NLR proteins, probably in parallel with NLR-associated transcription repressors, enabling potentially fast and robust transcriptional reprogramming upon pathogen recognition.
Lastly, the identification and functional study of MUSE6 revealed the crucial impact of N-terminal acetylation on the turnover of SNC1. Biochemical analysis uncovered that SNC1 undergoes alternative translation initiation, which provides respective substrates for NatA and NatB. SNC1 peptides translated from the first Met are targeted by NatA, and the acetylation serves as a degron, while SNC1 peptides initiated from the second Met are targeted by NatB, which stabilizes the protein. Different acetylation events on SNC1 are speculated to provide more flexibility to maintain its homeostasis.
Overall, my Ph.D thesis research contributes to the better understanding of NLR protein regulation and activation.Science, Faculty of
Botany, Department of
Graduate
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Dong, Xiaoou. "The roles of MUSE1 and MUSE15 in plant innate immunity." Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/58400.
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Mesmar, Joelle. "An investigation into the role of ubiquitination in plant immunity." Thesis, University of Glasgow, 2009. http://theses.gla.ac.uk/659/.
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Plants have developed elaborate defence mechanisms to protect themselves against pathogens. Recently, the ubiquitin-proteasome pathway has been proven to play important roles in regulating plant disease resistance. Previously, the tobacco (Nicotiana tabacum) ACRE276 and its Arabidopsis homolog AtPUB17 have been identified as E3 ligases that are positive regulators of the Cf-9/Avr9- and N/p50-elicited hypersensitive response (HR) in tobacco. In addition, AtPUB17 is required for the RPM1- and RPS4-mediated resistance responses in Arabidopsis. The identification of AtPUB17 signalling partners would allow us to understand the mode of action of AtPUB17 during plant defence. AtPOB1, a BTB/POZ-domain protein was isolated as an AtPUB17 interactor in a yeast-two-hybrid screen. The aim of this study was to confirm this interaction and to investigate the potential involvement of AtPOB1 in mediating disease resistance responses. The analysis of the Atpob1 knock out plants revealed a novel BTB/POZ protein implicated in plant defence. Atpob1 plants rapidly accumulated reactive oxygen species (ROS), induced the expression of pathogenesis related (PR) genes and developed spontaneous necrotic lesions upon infection with a virulent pathogen. AtPOB1 transcript and protein levels were induced by virulent Pseudomonas syringae. And transient overexpression of AtPOB1 in Cf-9 tobacco compromised the Avr9-triggered HR. In addition, Atpob1 plants showed signs of premature senescence. These results indicate that AtPOB1 is a negative regulator of plant defence- and senescence-associated pathways. The Nicotiana benthamiana AtPOB1 homolog was also identified and its cDNA sequence was used to investigate the role of NbPOB1 and its close relative NtPOB1 in disease resistance signalling. Transient overexpression of NbPOB1 and RNA interference (RNAi)-based silencing of NtPOB1 in Cf-9 tobacco compromised and accelerated the Avr9-triggered HR, respectively. Moreover, virus induced gene silencing (VIGS) of NbPOB1 accelerated the dark-induced senescence in N. benthamiana leaves. These observations identify NbPOB1 and NtPOB1 as the orthologs of AtPOB1. The subcellular localization of AtPOB1 and NbPOB1 was analyzed by transiently overexpressing GFP-AtPOB1 and GFP-NbPOB1 fusion proteins in tobacco leaf tissue. Analysis by confocal microscopy revealed that GFP fluorescence was localized in the nucleus of leaf tissue tested. The overexpression of AtPOB1 fused with a nuclear export signal (NES) failed to compromise the Avr9-triggered HR in Cf-9 tobacco, indicating the nuclear localization of AtPOB1 is crucial for its function. The BTB/POZ domain is a highly conserved protein-protein interaction interface that mediates homo- and/or hetero-dimerization of BTB/POZ proteins. The D146A and D141A mutation in the BTB/POZ domain of AtPOB1 and NbPOB1, respectively reduces their dimerization efficiency. These mutants fail to negatively regulate the Cf-9/Avr9-mediated HR, supporting the importance of an intact BTB/POZ interface for the function of AtPOB1 and NbPOB1. Finally, yeast-two-hybrid and immunoprecipitation assays indicate that AtPOB1 interacts with AtCUL3A, a component of E3 ligase complexes, in which AtPOB1 would confer substrate-specificity. We propose that AtPOB1 (and Nicotiana POB1) negatively regulate cell death and senescence possibly through ubiquitin-mediated pathways.
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Tkacz, Andrzej. "Plant genotype, immunity and soil composition control the rhizosphere microbiome." Thesis, University of East Anglia, 2013. https://ueaeprints.uea.ac.uk/48113/.
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Three model plant and three crop plant species were grown for three generations in sand and compost. Pots were inoculated with 10 % soil initially, and with 10% of growth medium from the previous generation in generations 2 and 3, keeping replicates separate for all three generations. The microbiome community structure of the plant rhizosphere in each generation was characterised using ARISA DNA fingerprinting and 454 sequencing. Rhizosphere bacterial and fungal communities are different from those in bulk soil and there are also differences in the microbial community between different plant species. Plants both select and suppress specific bacteria and fungi in the rhizosphere microbiome, presumably via composition of their root exudates. Two out of three most abundant bacteria selected in the rhizosphere were isolated. These isolates proved to possess plant growth promotion properties. Plants are able to “farm” the soil in order to enrich it with plant growth promoting rhizobacteria (PGPR) species. However, in some plant species rhizospheres, invasions of opportunists and pathogens take place, mimicking events in plant monocultures. Other experiments using this multi-replicate system allowed for statistical analysis of the influence of Arabidopsis and Medicago mutants on the rhizosphere microbiome. Three groups of Arabidopsis mutants were tested: plants unable to produce aliphatic glucosinolates, plants impaired in the PAMP-triggered immune response and plants unable and over-expressed in methyl halides production and one group of Medicago mutants which are impaired in the mycorrhization ability. All these plant genotypes, except those for methyl-halide production and one genotype involved in PAMP response, significantly altered the rhizosphere microbiome.
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Buxa, Stefanie Vera [Verfasser]. "Microscopic identification of plant immune responses in phloem tissue of higher plants relating to bacterial infection / Stefanie Vera Buxa." Gießen : Universitätsbibliothek, 2014. http://d-nb.info/1068773200/34.
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Muwanga, Catherine. "An assessment of Hypoxis hemerocallidea extracts, and actives as natural antibiotic, and immune modulation phytotherapies." Thesis, University of the Western Cape, 2006. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_3303_1184589097.
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In South Africa, the crude aqueous extract from Hypoxis hemerocallidea is used by AIDS patients to treat opportunistic infections, such as tuberculosis. The rapid emergence of multidrug-resistant tuberculosis, and extreme drug resistant tuberculosis, in recent years, is a major threat to human health. The treatment of TB, nosocomial bacterial infections, and fungal infections is now a clinical challenge, especially in the immuno-compromised individual. There is a dire need for novel antibiotic alternatives with phytotherapies and plant-derived compounds as potentially promising alternatives. The main objective of this study was to investigate the antimycobacterial activity of Hypoxis hemerocallidea, a South African medicinal plant, using Mycobacterium smegmatis.
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Yin, Chaoyan. "Deciphering the MtSymCRK signaling pathway controlling chronic infection during Medicago-Rhizobium symbiosis." Electronic Thesis or Diss., université Paris-Saclay, 2024. http://www.theses.fr/2024UPASB038.
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En conditions de carence azotée, les légumineuses peuvent établir une symbiose avec des bactéries fixatrices d'azote du sol (rhizobia) dans un nouvel organe racinaire, la nodosité, où les rhizobia fixent l'azote atmosphérique pour la plante. Une symbiose efficace nécessite des densités spectaculaires de bactéries différenciées fixatrices d'azote dans les cellules symbiotiques des nodosités. Malgré une colonisation bactérienne massive, les cellules symbiotiques ne présentent pas de réactions de défense apparentes, ce qui indique que l'état d'immunité du nodule est finement contrôlé pour permettre l'établissement et le maintien du partenaire symbiotique. Parmi les gènes de légumineuses identifiés impliqués dans ce processus, MtSymbiotic CYSTEINE-RICH RECEPTOR-LIKE KINASE (MtSymCRK) contribue à la répression de l'immunité du nodule. MtSymCRK code un récepteur kinase riche en cystéines, non arginine-aspartate (non-RD), appartenant à la famille des récepteurs riches en cystéines impliqués dans l'immunité des plantes. MtSymCRK est spécifiquement exprimé dans les cellules infectées des nodosités après l'internalisation des rhizobia et supprime les réactions de défense. Cependant, la voie de signalisation par laquelle MtSymCRK module l'immunité dans la nodosité pendant la symbiose n'est pas connue. Le projet de thèse a pour objectif d'étudier comment l'immunité des plantes est contrôlée dans les nodosités symbiotiques en décryptant les partenaires protéiques en aval de MtSymCRK impliqués dans la voie de signalisation lors d'une infection chronique chez Medicago littoralis. Nous avons identifié un partenaire interagissant avec MtSymCRK par une approche originale combinant un crible double hybride à haut débit en levure (Y2H) d'une banque d'ORFs d'Arabidopsis thaliana (A. thaliana) à de la génétique translationnelle vers Medicago truncatula (M. truncatula). Parmi les sept protéines identifiées chez A. thaliana interagissant avec le domaine kinase de MtSymCRK, AtGRF8 (GENERAL REGULATORY FACTOR 8) appartenant à la famille des 14-3-3, a été sélectionné comme étant le candidat le plus prometteur. Nous avons recherché l'homologue le plus proche chez M. truncatula, à savoir MtGRF8, et confirmé l'interaction entre le domaine kinase de MtSymCRK et MtGRF8.Afin de déterminer le rôle de MtGRF8 dans la symbiose, nous avons caractérisé Mtgrf8, un mutant d'insertion Tnt1 de Medicago littoralis. Nous avons montré que Mtgrf8 présente un dysfonctionnement symbiotique caractérisé par des réponses de défense et de sénescence qui compromettent la survie intracellulaire des rhizobia dans les nodosités. Enfin, basé sur l'hypothèse que certaines 14-3-3 et MtSymCRK régulent l'immunité et la production d'éthylène, nous avons également initié la recherche de protéines interagissant avec MtGRF8 par une approche Y2H.Dans son ensemble, ce travail contribue à déchiffrer la voie de signalisation de MtSymCRK au cours de l'infection chroniqueIn nitrogen-deficient conditions, legumes can establish a symbiosis with soil nitrogen-fixing bacteria (rhizobia) inside a new root organ, the nodule, where the rhizobia fix atmospheric nitrogen for the plant. Efficient symbiosis requires symbiotic nodule cells that host spectacular densities of nitrogen-fixing differentiated bacteria. Despite massive bacterial colonization, the symbiotic cells do not show apparent defense reactions, indicating that the nodule immune status is tightly controlled to allow the establishment and maintenance of the symbiotic partner. Among the identified legume genes involved in this process, MtSymbiotic CYSTEINE-RICH RECEPTOR-LIKE KINASE (MtSymCRK) contributes to the repression of the nodule immunity. MtSymCRK encodes a non-arginine-aspartate (non-RD) cysteine-rich receptor-like kinase belonging to the cysteine-rich kinase family of receptors involved in plant immunity. MtSymCRK is specifically expressed in nodule-infected cells after rhizobia internalization and prevents defense reactions. However, the signaling pathway by which MtSymCRK modulates the nodule immune response during symbiosis is not known. The PhD project aims to address how plant immunity is controlled in symbiotic nodules by deciphering the downstream MtSymCRK protein partners involved in the signaling pathway during chronic infection in Medicago littoralis. We identified an interacting partner of MtSymCRK using an original approach combining a high-throughput Yeast Two-Hybrid (Y2H) screen of an Arabidopsis thaliana (A. thaliana) open reading frames (ORFs) library and translational genetics towards Medicago truncatula (M. truncatula). Among seven A. thaliana identified proteins interacting with the kinase domain of MtSymCRK, AtGRF8 (GENERAL REGULATORY FACTOR 8) belonging to the 14-3-3 protein family has been selected as the most promising candidate. We searched for the closest homolog in M. truncatula, namely MtGRF8, and confirmed the interaction between the kinase domain of MtSymCRK and MtGRF8.To determine the role of MtGRF8 during symbiosis, we characterized Mtgrf8, a Medicago littoralis Tnt1 insertion mutant line. We showed that Mtgrf8 exhibits a symbiotic dysfunctioning characterized by defense and senescence responses that compromises the intracellular survival of the rhizobia in nodules. Finally, based on the hypothesis that some 14-3-3 and MtSymCRK regulate immunity and ethylene production. we also initiated the search for MtGRF8-interacting proteins by using a Y2H assay. Taken together, this work contributes to decipher the MtSymCRK signalling pathway during chronic infection
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Lian, Kehui. "Genetic analysis of anp2/3-mkk6-mpk4 cascade in plant immunity." Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/57798.
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Wu, Chih-Hang. "A complex NLR signalling network mediates immunity to diverse plant pathogens." Thesis, University of East Anglia, 2016. https://ueaeprints.uea.ac.uk/62253/.
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Both plants and animals rely on nucleotide-binding domain leucine-rich repeat-containing (NLR) proteins to respond to invading pathogens and activate immune responses. An emerging concept in NLR biology is that “sensor” NLR proteins are often paired with “helper” NLR proteins to mediate immune signalling. However, the degree to which NLRs form signalling networks beyond sensor and helper pairs is poorly understood. In this thesis, I discovered that a large NLR immune signalling network with a complex architecture mediates immunity to oomycetes, bacteria, viruses, nematodes, and insects. Helper NLRs in the NRC (NLR-required for cell death) family are functionally redundant but display distinct specificities towards diverse sensor NLRs. Several sensor NLRs, including Rx, Bs2 and Sw5b, signal via interchangeable NRC2, NRC3 or NRC4, whereas some other sensor NLRs have a more limited downstream spectrum. For example, Prf signals via interchangeable NRC2 or NRC3 but not NRC4, and Rpi-blb2 signals via only NRC4. These helper/sensor NLRs form a unique phylogenetic superclade, with the NRC clade sister to the sensor NLR clades. The network has emerged over 100 million years ago from an NLR pair that diversified into up to one half of the NLRs of asterids. I propose that this NLR network increases evolvability and robustness of immune signalling to counteract rapidly evolving plant pathogens.
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Cui, Beimi. "Identification and characterization of SNO regulated genes (SRGs) in plant immunity." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/15668.
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A conspicuous feature of plants responding to pathogen invasion is the synthesis of nitric oxide (NO), a redox signal. NO regulates protein function by S-nitrosylation, the addition of an NO moiety to a cysteine thiol to form an S-nitrosothiol. A key theme of NO function is reprogramming plant immune-related gene expression. However, it is still not clear how the NO signal is translated into transcriptional changes. Here we explored the potential role of a sub-group of SNO Regulated Genes (SRGs) uncovered by global expression profiling. Firstly, transgenic plants containing the SRG1 or SRG3 promoter fused to glucuronidase gene GUS together with qRT-PCR assays confirmed that transcripts of SRGs could be induced by NO and pathogen challenge, suggesting that SRGs may be involved in NO signalling related to plant immunity. More importantly, transient and stable overexpression of SRG genes induced hypersensitive response (HR)-like cell death development, which is often associated with pathogen effector-triggered immunity. Furthermore, transgenic plants constitutively expressing SRG genes exhibited enhanced ROS accumulation, PR1 transcript accumulation, and increased resistance to Pseudomonas syringae (Pst) DC3000 compared with Col-0 wild type plants. In contrast, lines with T-DNA insertions into SRG genes exhibited susceptibility to Pst DC3000. These data suggested SRGs act as the positive regulators in plant immunity. In order to further explore how NO regulates these SRGs in plant immunity, we focused on SRG1 and found SRG1 could be S-nitrosylated in vitro and in vivo. Moreover, electrophoretic mobility shift assays showed SRG1 could bind to an AGT motif and the transcriptional activity was blunted in the presence of NO, suggesting that the DNA binding activity of SRG1 is redox-modulated. Further, a transient repression activity assay showed that SRG1 has repression activity and this activity was impaired in the gsnor1-3 mutant, which has a high S-nitrosothiols level. These data suggested NO could block SRG1 transcriptional activity in vitro and in vivo. Furthermore when the SRG1 overexpression line was crossed with gsnor1-3 the SRG1-mediated resistance related phenotypes were suppressed. These data demonstrated NO negatively regulates SRG1 transcriptional activity during plant immunity. SRG1 may therefore be an important regulator of NO signalling and subsequent regulate transcription during plant immunity. Additionally, NO may negatively feedback to inhibit transcriptional activity of SRG1 to control its repression activity, to enable the activation of plant immunity.
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Jin, Lin. "The Bacterial AvrE-Family Type-III Effector Proteins Modulate Plant Immunity via Targeting Plant Protein Phosphatase 2A Complexes." The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1458339056.
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Naumenko, Anastasia Nikolayevna. "Dissection of Innate Immunity in Tomato and Tolerance to Bacterial Wilt in Solanaceae species." Thesis, Virginia Tech, 2013. http://hdl.handle.net/10919/19315.
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Unlike mammals, plants do not have specific immune cells. However, plants can still recognize pathogens and defend themselves. They do that by recognizing microbial-associated molecular patterns (MAMPs) and secreted pathogen proteins, called effectors. MAMP-triggered immunity (MTI) relies on recognition of MAMPs by leucine-rich repeats (LRRs) pattern-recognition receptors (PRRs). The best-studied LRR PRR is Flagellin-Sensitive 2 (Fls2), the receptor of a 22-amino acid long epitope of bacterial flagellin, called flg22. In this project, alleles of FLS2 of different tomato cultivars were sequenced and compared to each other to get insight into natural selection acting on FLS2 and to identify residues important for ligand binding. This information may be used in the future to engineer Fls2 for improved ability to recognize flagellin. MTI can be suppressed by effectors secreted by bacteria into plant cells through the type III secretion system. On the other hand, plants are equipped with repertoires of resistance proteins, which can recognize some pathogen effectors. If a pathogen carries an effector that is recognized, effector-triggered immunity (ETI) is activated and the plant is resistant. Here, eggplant breeding lines were screened for their ability to activate ETI upon recognition of effectors of the soil borne pathogen Ralstonia solanacearum, a causative agent of bacterial wilt. Four effectors were found to trigger plant defenses in some of the lines. This is the first step in cloning the genes coding for the responsible resistance proteins. These genes may be used in the future for engineering tomato and potato for resistance to bacterial wilt.Master of Science in Life Sciences
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Gully, Kay. "The plant immune system : induction, memory and de-priming of defense responses by endogenous, exogenous and synthetic elicitors." Thesis, Angers, 2019. http://www.theses.fr/2019ANGE0001/document.
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En tant qu’organismes sessiles, les plantes doivent réagir rapidement et intensément, via des réponses défensives, pour repousser les pathogènes invasifs. Le système immunitaire des plantes peut être déclenché par des molécules élicitrices exogènes ou endogènes. Une autre classe d’éliciteurs, les éliciteurs synthétiques, contient également des composés promouvant une réponse défensive.Dans ce manuscrit, je décris la découverte et caractérisation d’une nouvelle famille de petits peptides endogènes potentiellement sécrétés(PROSCOOP), dont les membres incluent de petits peptides (SCOOP). Je démontre que les SCOOP sont impliqués dans les mécanismes de défense de la plante et le développement racinaire. Une variété de peptides SCOOP induit des réponses défensives de courtes et longues durées.De plus, des traitements avec le peptideSCOOP12 induisent une résistance à Pseudomonas syringae chez Arabidopsis.Dans la seconde partie de cette thèse, je démontre que le traitement des plantes avec un éliciteur synthétique peut mener à une mémoire transcriptionnelle à long terme, et que le challenge subséquent des plantes traitées par application d’un éliciteur exogène désactive cette mémoire transcriptionnelle. En conclusion, ma thèse présente (1) la diversité des fonctions que peuvent avoir ces éliciteurs et (2) l’impact sur les systèmes de défense de la plante et ses conséquences sur la mémoire et le développement de la planteAs sessile organism, plants have to react quickly and strongly with defense responses to repel any invading pathogen. The plant immune system can be triggered by exogenous or endogenous elicitor molecules. Another class of elicitors are defense promoting compounds which are also known as synthetic elicitors. Here I describe the discovery and characterization of a novel family of potentially secreted small endogenous peptides (PROSCOOP) which members harbor small peptides (SCOOPs). I show that the SCOOP family is involved in plant defense and root development. Various SCOOP peptides induce short- and long-term defense responses. Moreover, treatments with the SCOOP12 peptide induce the resistance against Pseudomonas syringae in Arabidopsis. In the second part of this thesis, I show that treatments with a synthetic elicitor can lead to long-term transcriptional memory and that subsequent challenging of such plants with an exogenous elicitor reverted this transcriptional memory. In conclusion, my thesis shows (1) how diverse the function of these elicitors can be and (2) the impact the plant defense system and its triggers have on plant development and memory
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Ortiz, Diana. "Étude des bases moléculaires de la reconnaissance de l’effecteur fongique AVR-Pia par le récepteur immunitaire du riz RGA5." Thesis, Montpellier, SupAgro, 2016. http://www.theses.fr/2016NSAM0011/document.
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Les maladies des plantes causées par les champignons sont un problème majeur en agriculture. Pour les contrôler, les gènes de résistance (R) qui permettent de développer des variétés de plantes résistantes sont des éléments clés. La majorité des gènes R codent pour des protéines NLRs caractérisées par la présence d'un domaine de liaison aux nucléotides (NB-ARC) et un domaine de répétitions riches en leucines (LRR). Ces protéines agissent comme des récepteurs immunitaires intracellulaires et reconnaissent des facteurs de virulence des agents pathogènes appelés effecteurs. Les champignons phytopathogènes possèdent de vastes répertoires d'effecteurs qui contiennent centaines de protéines sécrétés, de petites tailles et sans similarités de séquence entre elles.La première question abordée dans ma thèse concerne l’origine de l'immense diversité des effecteurs fongiques. Une analyse structurale a identifié une famille d’effecteurs de séquences différentes mais qui possèdent une structure conservée. Cette famille a été appelée MAX-effectors (Magnaporthe Avrs and ToxB like) et elle est particulièrement importante chez Magnaporthe oryzae, l'agent causal de la pyriculariose du riz. Par des analyses d'expression, j'ai confirmé que la majorité des effecteurs MAX de M. oryzae sont spécifiquement exprimés durant la phase précoce de l'infection, suggérant une fonction importante durant la colonisation de la plante. Les effecteurs MAX constituent la première famille d'effecteurs fongiques définis par leur structure. Cette étude apporte donc de nouvelles pistes pour l'identification d'effecteurs chez les champignons et contribue à une meilleure compréhension de l'évolution des effecteurs. En effet, le scénario observé chez les effecteurs MAX suggère que beaucoup d’effecteurs fongiques appartiennent à un nombre restreint de familles d'effecteurs définies par leur structure. La seconde question que j’ai abordée durant ma thèse est le mécanisme moléculaire de la reconnaissance des effecteurs par les NLRs. J'ai abordé cette question en étudiant la reconnaissance de l'effecteur AVR-Pia par le couple de NLRs RGA4/RGA5. Des travaux précédents ont montré que RGA5 agit comme récepteur et se lie directement à AVR-Pia tandis que RGA4 agit comme élément de signalisation constitutivement actif, qui, en absence de l’agent pathogène, est réprimé par RGA5. Un domaine de RGA5, normalement absent chez les protéines NLR et similaire à la chaperonne du cuivre ATX1 (domaine RATX1), interagit physiquement avec AVR-Pia. Il a été suggéré que ce domaine RATX1 puisse agir comme un leurre de la cible de virulence d’AVR-Pia. Ce leurre, intégré dans la structure de RGA5, permettrait de « piéger » l’effecteur par interaction directe et jouerait donc un rôle crucial dans sa reconnaissance spécifique. Grâce à une analyse structurale détaillée d’AVR-Pia j’ai pu confirmer le rôle central de l'interaction AVR-Pia-RATX1 dans la reconnaissance de cet effecteur ce qui conforte le modèle du « leurre intégré ». De plus, j’ai caractérisé la surface d'interaction avec laquelle AVR-Pia lie le domaine RATX1. De plus, j'ai détecté des interactions entre AVR-Pia et d'autres parties de RGA5, indépendantes du domaine RATX1, notamment les domaines NB-ARC et LRR. Ceci a permis de développer un modèle qui explique comment la liaison d’un effecteur à un récepteur NLR comportant un leurre intégré par différentes interactions indépendantes conduit à une reconnaissance très sensible et spécifique qui est peu affectée par des mutations ponctuelles de l’effecteur. En résumé, cette étude a produit des connaissances nouvelles sur la fonction des récepteurs des plantes de type NLRs et sur leur capacité à reconnaitre des effecteurs. Ceci contribue à une meilleure compréhension du système immunitaire des plantes, ce qui est un élément important pour l’obtention de cultures durablement résistantes aux maladiesPlant diseases caused by fungi constitute a worldwide threat to food security and disease resistance (R) genes that allow to breed resistant crops are key elements for efficient disease control. The vast majority of R genes code for NLR multi domain proteins characterized by nucleotide-binding and leucine-rich repeat domains and acting as intracellular immune receptors for pathogen-secreted virulence factors termed effectors. Phytopathogenic fungi possess huge effector repertoires that are dominated by hundreds of sequence-unrelated small secreted proteins. The first question I addressed in my PhD thesis is: how is the tremendous diversity of fungal effectors generated? A structural analysis had identified the family of sequence-unrelated but structurally conserved MAX-effectors (Magnaporthe Avrs and ToxB like) that has expanded specifically in Magnaporthe oryzae the causal agent of rice blast disease. By expression analysis, I confirmed that the majority of M. oryzae MAX-effectors are expressed specifically during early infection suggesting important functions during host colonization. MAX effectors are the first structurally defined family of effectors in fungi and this study gives therefore news clues for the identification of candidate effectors in fungi and constitutes a crucial step towards a better understanding of effector evolution. In fact, the scenario observed for MAX-effectors leads to the hypothesis that the enormous number of sequence-unrelated fungal effectors belong in fact to a restricted set of structurally conserved effector families.The second question I investigated in my PhD thesis is: what are the molecular mechanisms of effector recognition by NLR immune receptors? I addressed this question by studying recognition of the M. oryzae effector AVR-Pia by the rice NLR pair RGA4/RGA5. Previous work has shown that RGA5 acts as a receptor that binds directly to AVR-Pia while RGA4 acts as a constitutively active signaling protein that is, in the absence of pathogen, repressed by RGA5. This functional interaction involves formation of an RGA4/RGA5 receptor complex. By protein-protein interaction studies, I showed that complex formation involves interactions between the RA4 and RGA5 NB-ARC and LRR domains, in addition to previously identified interactions between the coiled-coil domains. AVR-Pia recognition seems not to induce dissociation of the RGA4/RGA5 complex but a ternary RGA4/RGA5/AVR-Pia complex could also not be detected consistently. How effector recognition is translated into receptor complex activation remains therefore to be elucidated in more detail in the future. Previous work has shown that a domain of RGA5 normally not present in NLRs and related to the copper chaperone ATX1 (RATX1 domain) interacts physically with AVR-Pia and may be crucial for effector recognition. The RATX1 domain was hypothesized to mimic the true host targets of AVR-Pia leading to the development of the ‘integrated decoy’ model that states that unconventional domains in NLRs act as decoys in the recognition of effector proteins. By detailed structure-informed analysis of AVR-Pia, I could confirm the pivotal role of the AVR-Pia-RATX1 interaction for effector recognition lending important support to the integrated decoy model. In addition, I could precisely characterize the interaction surface with which AVR-Pia binds to the RGA5 RATX1 domain. Finally, I detected interactions of AVR-Pia with other parts of RGA5, in particular the NB-ARC and the LRR domains. Based on these results, I developed a model that explains how such binding to several independent sites in NLRs leads to high overall affinity and robust effector recognition that is resilient to effector mutations. Taken together, this study provides important novel insight into NLR function and effector recognition and contributes by this to a better understanding of plant immunity which is crucial for generating durable disease resistance in crops
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Shepherd, Ryan William. "PHYLLOPLANINS: NOVEL ANTIFUNGAL PROTEINS ON PLANT LEAF SURFACES." UKnowledge, 2010. http://uknowledge.uky.edu/gradschool_diss/763.
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Secreted surface proteins are an innate immune defense component employed by animals to inhibit invading microbes. Surface proteins have not been documented in plants, even though the aerial leaf surface, or phylloplane, is a major site of pathogen ingress. We have discovered novel proteins, termed phylloplanins, which accumulate on leaf surfaces of Nicotiana tabacum, and we have isolated the gene Phylloplanin that is unique in gene databases. Natural and E. coli-expressed phylloplanins inhibit spore germination and limit leaf infection by the oomycete pathogen Peronospora tabacina.
We investigated the site of phylloplanin biosynthesis using biochemical techniques. These techniques included radiolabeling of detached trichome glands, radiolabeling of epidermal peels, analysis of leaf water washes of various Nicotiana plants, and examination of guttation fluid, leaf vein contents, and extracellular fluid. From these experiments, we tentatively conclude that phylloplanins are produced by hydathodes, or an unknown surface secreting system, but not by glandular secreting trichomes. Future experiments with the phylloplanin promoter, whose elucidation is described herein, and its fusion to a reporter gene (GUS or GFP), will undoubtedly provide further insight into the location of phylloplanin biosynthesis and deposition. We suggest that the hydrophobic nature of phylloplanins aids in their dispersal over the leaf surface.
Phylloplanins constitute a first-point-of-contact, rapid response, innate immune deterrent to pathogen establishment on N. tabacum leaf surfaces, and are the first studied representatives of a novel protein class in the plant kingdom. Further study of leaf surface proteins is justified to understand further their roles in plant defense, and to investigate their potential in agricultural biotechnology.
Additionally, we describe miscellaneous observations we have made during the course of this research. Low molecular mass proteins (as yet uncharacterized) are washed from leaf surfaces of sunflower, soybean, and other plants. Pathogenesis-related (PR-)-5a, a known antifungal protein, was found to be present on the leaf surfaces of healthy plants, although its function there remains unknown. A phylloplanin homologue from Arabidopsis appears to be antibacterial. Further study of this protein is warranted. We note that proteins can also be recovered from N. tabacum root surfaces, or the rhizoplane, but we have not further characterized these proteins.
In summary, novel surface-accumulated proteins, termed phylloplanins, and the gene encoding these have been discovered in N. tabacum. An antifungal function for phylloplanins is reported, and evidence was found for a unique mechanism of surface deposition.
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Li, Xinyan. "Interplay between bacterial virulence and plant innate immunity in Ppseudomonas-arabidopsis interactions." Diss., Manhattan, Kan. : Kansas State University, 2006. http://hdl.handle.net/2097/243.
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Tong, Meixuezi. "Identification and analysis of E3 ligases and helper NLRs in plant immunity." Thesis, University of British Columbia, 2016. http://hdl.handle.net/2429/57683.
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Plant immunity is usually initiated with two types of immune receptors: 1) pattern recognition receptors (PRRs) recognize the conserved molecular features of pathogens (pathogen-associated molecular patterns, PAMPs) and trigger PTI (PAMP-triggered immunity) and; 2) nucleotide-binding/leucine-rich repeats (NLRs) serve as intracellular immune receptors with the ability to recognize the presence of relatively diverse pathogen effectors and trigger ETI (effector-triggered immunity). The Arabidopsis thaliana mutant snc1 contains a gain-of-function mutation in a Toll/interleukin-1 (TIR)-type NLR (TNL) gene and displays a dwarf morphology. Here, I report on the results of a snc1-influencing plant E3 ligase reverse genetic (SNIPER) screen that looked for snc1 plants with altered dwarfism in the presence of overexpressed E3 ligases. Six SNIPER genes were identified with four snc1-suppressors and two snc1-enhancers. SNIPER1/2/3 were selected for further characterization. The analysis of SNIPER1/2 is incomplete, thus is not included in this thesis.
Chapter 3 describes SNIPER3, previously known as SAUL1 (Senescence-Associated E3 Ubiquitin Ligase 1) or PUB44 (Plant U-box 44), which encodes a U-box-type E3 ligase. Our data suggests that SAUL1 plays a dual role in plant immunity: on one hand, SAUL1 positively regulates basal resistance; on the other hand, SAUL1 suppresses a typical TNL immune receptor SUSA1 (Suppressor of saul1) to prevent its autoimmunity.
ADR1, ADR1-L1 and ADR1-L2 are three homologous coiled-coil (CC)-type NLRs (CNLs), which were previously shown to work as helper NLRs. Chapter 4 further explores the specificity of the genetic requirement of ADR1s for typical TNLs, SNC1 and CHS2 (CHILLING SENSITIVE 2). Among the three ADR1 members, ADR1 is the leading contributor while ADR1-L1 is the least. Moreover, loss-of-function mutation of ADR1-L1 leads to over compensation of the transcript expression level of ADR1 and ADR1-L2 and results in the enhancement of snc1-mediated immunity.
Overall, the studies I completed as part of my Ph.D. thesis expand our knowledge of the roles of E3 ligases and ADR1s in plant defense and help us to better understand the sophisticated regulatory mechanisms of plant innate immunity.Science, Faculty of
Botany, Department of
Graduate
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Xu, Fan. "Functional analysis of a plant metacaspase in negative regulation of innate immunity." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/61596.
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Moore, John Wallace. "Foundation technologies in synthetic biology : tools for use in understanding plant immunity." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/6225.
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The plant hormone salicylic acid (SA) is an essential activator of plant immune responses directed against biotrophic pathogens. The transcription cofactor NPR1 (Nonexpressor of pathogenesis- related (PR) genes 1) functions to transduce the SA signal into an operational response directed to limited pathogen damage. In the absence of pathogen, NPR1 protein resides in the cytoplasm as a large molecular weight oligomer held together by disulphide bonding. Initiation of defence signalling leads to changes in intracellular redox conditions that promote NPR1 momomer release. Translocation of monomeric NPR1 to the nucleus results in the activation of over 2200 immune-related genes in Arabidopsis. NPR1 lacks a canonical DNA-binding domain but is known to perform part of its regulatory function through engagement of TGA factors (bZIP transcription factor). Induction of SA-dependent signalling is invariably associated with PR-1 gene expression and accumulation of mRNA for this gene serves as a useful marker of defence activation. However, both functional redundancy and stochastic factors limit the effectiveness of standard genetic approaches used in plant research, and thus much of the hierarchal processes surrounding NPR1-dependent gene activation are not fully understood. Using a synthetic biology approach we aim to complete exploratory work and set the foundations for the development of a yeast tool that can be used to manipulate and subsequently understand NPR1 function in relation to interacting partners and gene activation. Accordingly, using this tool we sought to create a conceptual protein circuit based on theoretical plant immunity. In completing this work we have developed a Saccharomyces cerevisiae strain that exhibits a highly oxidising intracellular redox environment. This was achieved by knocking out genes encoding S-nitrosoglutathione reductase (SFA1), flavohemoglobin (YHB1) and YAP1 (bZIP transcription factor), all important components in regulating cellular redox homeostasis and protein S-nitrosylation state in S. cerevisiae. Characterisation of this cell (designated Δsfa1yap1yhb1) reveals a high tolerance to such redox perturbations. Importantly, NPR1 is by default, assembled predominantly in the oligomeric form in this biological chassis. By activating two inducible inputs in the form of Arabidopsis S-nitrosoglutathione reductase (AtGSNOR) and Thioredoxin (AtTRXh5) which both function to promote NPR1 monomerisation, we have created a switch to selectively control NPR1 oligomer-monomer equilibrium. To complete the synthetic circuit, TGA3 was included, along with a modified yeast MEL1 promoter that has been customised to contain the TGA-responsive upstream activation sequence (termed the as-1 element) present in the promoter region of the PR-1 gene. Using FRET tools we were able to confirm nuclear interaction between monomeric NPR1 and TGA3, with this association appearing to induce as-1 element binding. However this process is not sufficient to activate a Luciferase (LUC) reporter gene, even when the GAL4 activation domain (GAL4 AD) is fused to NPR1. Ordinarily, a CUL3-dependent proteolysis-coupled transcription cycle is necessary to maintain efficient NPR1-dependent gene transcription in Arabidopsis. Although S. cerevisiae encodes an evolutionarily related CUL3 ortholog, examination by western blot demonstrates that NPR1 protein is stable in this cell, indicating an endogenous mechanism to degrade NPR1 is either not present or not functional in yeast. As such, this synthetic yeast tool represents a completely novel approach to identify missing components functioning in NPR1-mediated transcriptional regulation. Furthermore, in collaboration with a skilled bioinformatician, and using a rule-based stochastic modeling tool known as Kappa, we have been able to develop, for the first time, a preliminary mathematical simulation representative of NPR1-dependent gene activation that can be used as a foundation for future works.
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Pfeilmeier, Sebastian. "Elicitation and evasion of plant innate immunity by beneficial and pathogenic bacteria." Thesis, University of East Anglia, 2017. https://ueaeprints.uea.ac.uk/66876/.
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Plasma membrane-localized pattern recognition receptors (PRRs) are central components of the plant innate immune system. PRRs perceive characteristic microbial features, termed pathogen-associated molecular patterns (PAMPs), leading to pattern-triggered immunity (PTI). As PAMPs from both pathogenic and beneficial bacteria are potentially recognized, both must employ strategies to evade and/or suppress PTI. Here, I show that exopolysaccharides (EPS) and flagella-driven motility, both of which are regulated by the secondary messenger cyclic-di-GMP, are important virulence factors at different stages during Pseudomonas syringae pv. tomato (Pto) DC3000 infection of Arabidopsis thaliana. High levels of cyclic-di-GMP impaired flagellin accumulation in different Pseudomonas species, and helped bacteria to evade recognition by the PRR FLAGELLIN SENSING 2. In this case, immune evasion was fully explained by the effect of cyclic-di-GMP on flagellin synthesis rather than on EPS production. Nevertheless, an EPS-deficient Pto DC3000 mutant, Δalg/psl/wss, showed compromised virulence, and a combination of two types of EPS appeared to be required for optimal in planta proliferation. In a complementary project, I tested whether PAMP recognition affects the interaction between the legume Medicago truncatula and its symbiotic partner Sinorhizobium meliloti. I transferred the PRR EF-TU RECEPTOR (EFR) from A. thaliana to M. truncatula, conferring recognition of the bacterial EF-Tu protein. Expression of EFR protected M. truncatula against the root pathogen Ralstonia solanacearum without compromising the overall symbiotic interaction with nitrogen-fixing S. meliloti. My results indicate that rhizobium either avoids PAMP recognition, or actively suppresses immune signalling during the infection process. Finally, I engineered a PAMP in S. meliloti by replacing the eliciting inactive flg22 epitope (derived from flagellin) with an eliciting epitope. My results suggest that legumes can be engineered with novel PRRs, as a biotechnological approach for broad-spectrum disease resistance, without perturbing the nitrogen-fixing symbiosis. Overall, my work contributes to our understanding of the molecular interaction between plants and bacteria.
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Palma, Kristoffer. "Regulation of plant innate immunity: the role of protein import and the novel MOS4-associated complex." Thesis, University of British Columbia, 2007. http://hdl.handle.net/2429/250.
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Plants have evolved sophisticated defence systems against pathogen infection. Initiation of induced defence signalling often involves specific recognition of invading pathogens by the products of specialized host Resistance (R) genes. Consequently, the pathogen is stopped at the site of infection. A unique dominant mutant in Arabidopsis thaliana, snc1, constitutively expresses pathogensis-related (PR) genes and exhibits enhanced resistance to bacterial and oomycete pathogens. SNC1 encodes an R-gene – a single amino acid change renders this protein constitutively active without interaction with pathogens. snc1 displays a stunted phenotype that may be caused by both the accumulation of toxic compounds and energy squandered on unnecessary defence instead of normal growth. The distinctive morphological phenotype of snc1 is intimately associated with the other resistance phenotypes, and provides a robust genetic tool for dissecting the signalling events downstream of snc1.
To identify genes important for defence signalling, we carried out a suppressor screen to identify modifier of snc1 (mos) mutants that restore the wild type size and morphology in the snc1 background. Furthermore, in most cases, a loss of sneakiness in mos mutants correlated with a reduced or abolished constitutive PR gene expression, SA accumulation and pathogen resistance in snc1 plants. These loss of function mutants represent defects in positive regulators of the snc1 pathway. I cloned and characterized two mos mutants, and showed that they both have roles in Arabidopsis innate immunity as well.
mos6 partially suppresses snc1 and exhibits enhanced disease susceptibility (EDS) to an oomycete pathogen. MOS6, identified by map-based cloning, encodes an alpha-importin subunit, one of 8 found in Arabidopsis, and has a demonstrated role in nucleocytoplasmic partitioning (protein import). Two other genes cloned by others from this screen, MOS3 and MOS7, encode components of the nuclear pore complex, implicating nuclear trafficking as a key regulator in plant innate immunity.
mos4 exhibits EDS to virulent and avirulent bacterial and oomycete pathogens. There is evidence that MOS4-mediated resistance is independent of the signalling protein NPR1. MOS4 encodes a protein with homology to human Breast Cancer Amplified Sequence 2 and with predicted protein-protein interaction domains. Subcellular localization of MOS4-GFP shows that MOS4 is localized to the nucleus. To illuminate the biochemical function of MOS4, a yeast-2-hybrid screen was conducted. One MOS4-interactor was a putative myb transcription factor, MOS4-Associated Complex Protein 1 (MAC1), also known at AtCDC5. MAC1 interacts directly with MOS4 in vitro and in planta. mac1 insertional mutants exhibit defects in immune responses to pathogens similar to that of mos4. In addition, mac1 also partially suppressed snc1 morphology and enhanced resistance.
Both MOS4 and MAC1 have homologs in humans and fission yeast that are members of a discrete protein complex that has been implicated in several different biological processes including RNA splicing, apoptosis and protein degradation. Using proteomics data from yeast and human, we found genes with homology to additional components of the orthologous complex in Arabidopsis, and isolated insertion mutants in these. Mutations in PRL1, which encodes a WD protein, display similar disease phenotypes to that of mos4 and mac1. AtCDC5 has DNA binding activity, suggesting that this complex may regulate defence responses through transcriptional control. Since the complex components along with their interactions are highly conserved from fission yeast to Arabidopsis and human, they may also have a yet-to-be identified function in mammalian innate immunity.
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Seck, Amadou. "Gene expression and signaling in Rxo1 governed innate immunity in cereals." Diss., Manhattan, Kan. : Kansas State University, 2008. http://hdl.handle.net/2097/1107.
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Huang, Yan. "Dissecting negative regulation of plant immunity through studying muse (mutant, snc1-enhancing) mutants." Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/45667.
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Pardal, Bermejo Alonso Javier. "Exploring the role of histone marks and chromatin remodelling ATPases in plant immunity." Thesis, University of Warwick, 2017. http://wrap.warwick.ac.uk/104239/.
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Plant cells require considerable transcriptional reprograming to mount an effective response to pathogens. Plant responses to pathogens have to be finely balanced with other vital biological processes such as development and growth. A major mechanism controlling the modulation of gene expression is chromatin remodelling. Chromatin remodelling requires histone covalent modifications and/or the action of ATP- dependent remodelling complexes. The combined action of these determine the accessibility of transcription factors and the basal transcription machinery to DNA and therefore greatly impact gene expression. There are several examples of histone modifying and chromatin remodelling enzymes previously shown to regulate plant development and immunity. This thesis explored the role of chromatin in plant defences, and how chromatin remodelling forms an integral part of the defence response. Chapter 1 aimed to discover a “hidden” signal of chromatin marks in plant defence-responsive genes using an array of bioinformatics techniques. Subsequently, histone H3K27 tri- methylation (H3K27me3) was identified as a mark associated with gene repression at defence-related loci. The role of histone H3K27me3 and its associated histone demethylase enzymes REF6 and ELF6 were empirically characterised. Chapter 2 is dedicated to a reverse genetics screening investigating the role of the chromatin remodelling ATPases Arabidopsis family in plant defences, and describes the most prominent phenotypes. And lastly, Chapter 3 dissects in greater detail the role of the chromatin remodelling ATPase EDA16 in plant defence. Pathogen assays, RNA-seq and other molecular techniques suggest that EDA16 is a negative regulator of immunity induced upon pathogen perception to regulate the amplitude of defence responses.
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Clarke, Christopher R. "Elucidating three novel mechanisms of Pseudomonas syringae pathogenicity." Diss., Virginia Tech, 2012. http://hdl.handle.net/10919/37378.
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Pseudomonas syringae is an important bacterial plant pathogen that, as a species, is known to cause disease on hundreds of different plant species. However, any individual pathovar of P. syringae typically only causes disease on one or a few plant species, which constitute the host range of the pathovar. Plants are generally resistant to most pathogens primarily because the plant innate immune system is capable of recognizing conserved microbial-associated molecular patterns (MAMPs). Adapted pathovars of P. syringae secrete effector proteins through a Type Three Secretion System (T3SS) to suppress the immune response elicited by their MAMPs. However, secretion of effectors can also trigger a strong plant immune response if the plant harbors resistance proteins capable of recognizing the secreted effectors. Successful pathovars, therefore, must secrete a combination of effectors capable of suppressing MAMP/Pattern-Triggered Immunity (PTI) without eliciting Effector-Triggered Immunity. Here we identify several novel strategies employed by P. syringae to overcome the plant immune system and cause disease. First, we demonstrate that, in place of the canonical T3SS used by all known pathogens of P. syringae, several apparently nonpathogenic isolates of P. syringae employ a novel T3SS that is functional but not necessary for colonization of plants. Despite being closely related to pathogenic isolates of P. syringae, the isolates employing the noncanonical T3SS do not cause disease on any tested plants and instead appear to act more as commensal organisms. Second, we advance the understanding of PTI by identifying a second region of bacterial flagellin that triggers PTI in addition to the archetypical MAMP flg22, which is recognized by the archetypical plant receptor FLS2. This new elicitor, termed flgII-28, is also detected by FLS2 and appears to be under selection in very closely related lineages of P. syringae. Alleles of flagellin present in one recently expanded and agriculturally problematic lineage of P. syringae appear to trigger less PTI on their host plant, tomato, than the ancestral allele suggesting that avoidance of PTI through allelic diversity in MAMPs is an effective alternative strategy to suppression of PTI through delivery of effectors. Finally, we start to elucidate a role for chemotaxis (chemical-directed movement) in P. syringae pathogenicity. Not only is chemotaxis required for pathogenicity of P. syringae on plants, but it also appears to contribute to delimiting the host range of several P. syringae pathovars. These results highlight that additional aspects of P. syringae pathogenicity, such as chemotaxis, can directly contribute to defining the host range of individual P. syringae pathovars. The current paradigm of P. syringae pathogenicity posits that MAMPS and the repertoire of effector proteins are the primary determinant of the host range of any P. syringae pathovar; in contrast these results inspire a more nuanced view of pathogenicity that considers multiple aspects of the infection process.Ph. D.
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Kosaka, Ayumi. "Studies on postinvasive resistance of Arabidopsis thaliana against multiple fungal pathogens." Kyoto University, 2019. http://hdl.handle.net/2433/245323.
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Kyoto University (京都大学)0048
新制・課程博士
博士(農学)
甲第22128号
農博第2374号
新制||農||1073(附属図書館)
学位論文||R1||N5236(農学部図書室)
京都大学大学院農学研究科応用生物科学専攻
(主査)教授 髙野 義孝, 教授 田中 千尋, 教授 寺内 良平
学位規則第4条第1項該当
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Wang, Jingyu. "Transcriptional control of immune-responsive genes by DNA methylation and demethylation and its relevance in antibacterial defense." Thesis, Paris 6, 2017. http://www.theses.fr/2017PA066402.
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La méthylation et déméthylation de l'ADN jouent un rôle majeur dans la stabilité des génomes, l'empreinte génomique, la paramutation et le développement. En revanche, le rôle de cette régulation épigénétique a été peu étudiée dans les interactions hôtes-pathogènes. Dans ce projet de thèse, nous avons tout d'abord montré que la méthylation de l'ADN régule négativement la résistance d'Arabidopsis thaliana à une souche de Pseudomonas syringae pathogène. Nous avons également identifié un grand nombre de gènes de l'immunité ciblés directement par la méthylation de l'ADN dirigée par petits ARN dans leurs régions promotrices. Nous proposons que cette régulation génique permettrait de maintenir une faible expression basale de ces gènes et d'éviter ainsi des effets délétères qui seraient causés par une expression constitutive de la réponse immunitaire. De plus, nous montrons que la déméthylase active REPRESSOR OF SILENCING 1 (ROS1) facilite l'activation transcriptionnelle de gènes de l'immunité en laissant potentiellement des éléments de régulation en cis accessibles à des facteurs de transcription. Nous avons également démontré que ce facteur contribue à la résistance à P. syringae chez Arabidopsis, caractérisant ainsi la première déméthylase eucaryote dans la résistance antibactérienne. Sur la base de ces résultats, nous proposons que la méthylation de l'ADN maintient une faible expression basale de gènes de l'immunité en absence de pathogène, tandis que la déméthylation active assure une induction rapide de ces gènes au cours de la réponse immunitaire en favorisant potentiellement le recrutement de facteurs de transcription sur la chromatineDNA methylation and demethylation are regulatory processes involved in genome stability, genomic imprinting, paramutation and development. Until recently, very little was known about the role of these epigenetic processes in plant disease resistance and in the transcriptional control of immune-responsive genes. Here we provide evidence that DNA methylation negatively regulates antibacterial resistance against a virulent Pseudomonas syringae strain in Arabidopsis. Accordingly, we have identified a subset of defense genes that are targeted and repressed by RNA-directed DNA methylation (RdDM), presumably to prevent trade-off effects that would be caused by their constitutive expression and/or sustained induction. In addition, we found that the active DNA demethylase facilitates the transcriptional activation of some of these defense genes by pruning DNA methylation at their promoter regions and leaving cis-elements accessible for transcription factor binding. In addition, we show that the active demethylase REPRESSOR OF SILENCING 1 (ROS1) positively regulates late immune responses including Pathogen Associated Molecular Pattern (PAMP)-triggered callose deposition and salicylic acid (SA)-dependent defense response. We also demonstrate that ROS1 restricts Pto DC3000 propagation in Arabidopsis leaf secondary veins, providing the first example for a role of an active DNA demethylase in antibacterial resistance. Based on these findings we propose that DNA methylation maintains a low basal expression of some immune-responsive genes in normal growth condition, while active DNA demethylation ensures a rapid and pervasive induction of these genes upon bacterial pathogen detection
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Gao, Fang. "Arabidopsis MKK6 functions in parallel with MKK1 and MKK2 to negatively regulate plant immunity." Thesis, University of British Columbia, 2017. http://hdl.handle.net/2429/61324.
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Zhang, Fei [Verfasser]. "Functional analysis of Arabidopsis thaliana matrix metalloproteinases and MORC in plant immunity / Fei Zhang." Gießen : Universitätsbibliothek, 2016. http://d-nb.info/1110616457/34.
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Kaster, Margaux [Verfasser], and Sascha [Akademischer Betreuer] Laubinger. "The different levels of gene regulation in plant immunity / Margaux Kaster ; Betreuer: Sascha Laubinger." Tübingen : Universitätsbibliothek Tübingen, 2019. http://d-nb.info/1198859024/34.
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Benezech, Claire. "Développement et étude de systèmes d'interactions tripartites, légumineuses-rhizobia-pathogènes." Thesis, Toulouse 3, 2019. http://www.theses.fr/2019TOU30289.
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L'azote est un élément essentiel au développement des êtres vivants. Bien qu'il soit présent en grande quantité dans l'air, sous la forme de diazote, il n'est pas directement assimilable par la plupart des êtres vivants. Les légumineuses, par exemple, n'ont pas la capacité de l'assimiler sous cette forme. Cependant, dans un environnement carencé en azote, les plantes sont capables d'interagir avec des bactéries du sol, les rhizobia, qui sont des microorganismes fixateur d'azote atmosphérique grâce à un complexe enzymatique, la nitrogénase. En effet, ces bactéries vont réduire le diazote en ammonium, qui est assimilable par la plante. Les plantes hébergent ces bactéries dans des organes particuliers au niveau de leurs racines, les nodosités, où elles vont leurs fournir des nutriments. La plante tolère au sein de ses propres cellules une quantité importante d'organismes étrangers, environ un milliard par nodosité. La colonisation bactérienne massive des nodosités est permise grâce à la suppression des réponses immunes de la plante. Les racines sont en contact avec l'abondante flore microbienne du sol, ce qui soulève la question des conséquences liées à la potentielle vulnérabilité des organes symbiotiques ainsi que les plantes nodulées. L'objectif du projet de thèse était d'évaluer la vulnérabilité des organes symbiotiques. Pour cela, nous avons mis en place deux systèmes tripartites impliquant la légumineuse-modèle, Medicago truncatula, son symbionte, Sinorhizobium medicae et séparément deux microorganismes phytopathogènes, une bactérie Ralstonia solancearum et un champignon Sclerotinia sclerotiorum. Nous avons aussi caractérisé les réponses des nodosités face à ces deux pathogènes et cela en prenant les racines comme référence. Enfin, nous avons estimé l'influence de la nodulation et de la fixation d'azote sur la vulnérabilité des plantes face à l'agent pathogène bactérien. Les travaux effectués durant les trois ans de thèse indiquent que les nodosités sont des sites d'infection pour les agents pathogènes et qu'elles sont capables de répondre à la présence de pathogènes, néanmoins de manière différente et plus faiblement que les racines. Les résultats obtenus en utilisant l'un de nos systèmes tripartites suggèrent que la nodulation et la fixation d'azote peuvent conférer une plus grande sensibilité face aux agents pathogènesNitrogen is essential element for the development of all living beings. Although it is found in large quantity in the air, in the form of dinitrogen, it is not directly assimilable by most organisms. For example, plants are not able to assimilate this form. However, in a nitrogen deficient environment, legumes are able to interact with soil borne bacteria, rhizobia, which fix nitrogen thanks to an enzymatic complex, the nitrogenase. Indeed, bacteria reduce dinitrogen in ammonium; plants can assimilate this form. Plants host these bacteria in particular organs at the root level, the nodules, where they provide nutrients to bacteria. Plant tolerates in its own cells a tremendous quantity of foreign organisms, estimated to one billion of rhizobia per nodule. The massive bacterial colonization of nodules is allowed thanks to the repression of plant immunity. Roots are in contact with the abundant soil microbiota, which raises the question of the potential vulnerability of the symbiotic organs and nodulated plants. The phD project aimed to evaluate the nodules vulnerability. To achieve this, we set up two tripartite systems involving the model legume, Medicago truncatula, its symbiont, Sinorhizobium medicae and separately two phytopathogenic microorganisms, a bacterium, Ralstonia solancearum and the fungus, Sclerotinia sclerotiorum. We also characterized nodules responses to both pathogens using roots as reference. Finally, we estimated the influence of nodulation and nitrogen fixation on the plant vulnerability to pathogens. Work performed during these three years indicates that nodules are infection sites for pathogens. Those nodules are able to perceive the pathogen however, their response is different and less intense than that of roots. Results obtained with one of our tripartite system suggest that nodulation and nitrogen fixation give a greater sensitivity to pathogens
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Anderson, Ryan Gabriel. "Identification and functional characterization of RXLR effector proteins that are conserved between downy mildew pathogens and Phytophthora species." Diss., Virginia Tech, 2011. http://hdl.handle.net/10919/77204.
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Diverse pathogens secrete effector proteins into plant cells to manipulate host cellular processes. The genome of Hyaloperonospora arabidopsidis (Hpa), the causative agent of downy mildew of Arabidopsis, contains at least 134 candidate RXLR effector genes. These genes contain an RXLR motif required for effector entry into host cells. Only a small subset of these candidate effectors is conserved in related oomycetes. Here, we describe a comparative functional characterization of the Hpa RXLR effector HaRxL96 and a homologous gene, PsAvh163, from the soybean pathogen Phytophthora sojae. HaRxL96 and PsAvh163 are induced during early stages of infection and carry a functional RXLR motif that is sufficient for protein uptake into plant cells. Both effectors can suppress or activate immune responses in soybean, Nicotiana, and Arabidopsis. Several SA-responsive defense genes are suppressed in Arabidopsis Col:HaRxL96 and Col:PsAvh163 during an incompatible interaction with Hpa Emoy2. Both effectors are localized to the nucleus and cytoplasm of plant cells. Nuclear localization of both effectors is required for proper virulence functions, including suppression of basal resistance and RPP4-mediated immunity to virulent and avirulent Hpa, respectively. In addition, both effectors interact with plant U-box (PUB) proteins that are conserved between diverse plant species. The targeted PUB proteins are negative regulators of plant immunity in Arabidopsis. These experiments demonstrate that evolutionarily-conserved effectors from different oomycete species can suppress immunity in plant species that are divergent from the source pathogen's primary host.Ph. D.
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Bhardwaj, Vaibhav. "Keeping time on the plant-pathogen arms race : a role for the plant circadian clock in immune response." Master's thesis, University of Cape Town, 2011. http://hdl.handle.net/11427/10104.
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In this study, Arabidopsis thaliana (Arabidopsis) in the Columbia-0 (Col-0) background showed time-of-day variation in susceptibility to the plant-pathogen Pseudomonas syringae DC3000 pathovar tomato (P. syringae DC3000) when infected under constant light and temperature conditions. Wild type plants showed least susceptibility at circadian time (CT) 26 and 50, which correspond to "subjective" morning. Plants were most susceptible when infected at CT42 and CT66, "subjective" night.