The Arabidopsis TGA family of basic leucine zipper transcription factors regulate the expression of pathogenesis-related genes and are required for resistance to disease. Members of the family possess diverse properties in respect to their ability to transactivate and interact with NPR1, the central regulator of systemic acquired resistance in Arabidopsis. Two TGA factors, TGA1 and TGA2, have 83 % amino acid similarity but possess differing properties. TGA1 does not interact with NPR1 but is able to transactivate, while TGA2 interacts with NPR1 but is unable to transactivate. This study uses these two TGA factors to identify amino acids that are responsible for their function. <p>Four cysteines residues within TGA1 were targeted for study by site-directed mutagenesis and the resulting mutants were tested for interaction with NPR1 in yeast. The construct containing a mutation of cysteine 260 (Cys-260) interacted well with NPR1, while those with mutations at Cys-172 or Cys-266 interacted poorly. The Cys-260 mutant also displayed the greatest decrease in transactivation potential in yeast, while mutation of Cys-172 or Cys-266 resulted in smaller decreases. Mutation of Cys-287 had no effect on NPR1 interaction or transactivation. Combining various point mutations in a single protein did not increase NPR1 interaction or transactivation levels, indicating that Cys-260 is crucial for regulating TGA1 properties. Cysteines possess the unique ability of forming reversible disulfide bonds which have been shown to regulate several mammalian cellular processes. The observation that mutation of a single TGA1 cysteine (Cys-260) greatly alters the proteins properties provides a convincing argument that oxidoreduction of this residue is important for its regulation, possibly through the formation of a disulfide bond with either Cys-172 or Cys-266. <p>To test whether other members of the TGA family could be regulated by oxidoreduction, several TGA2 constructs were created that introduced Cys at positions corresponding to those found in TGA1. When tested in yeast none were able to transactivate but continued to interact with NPR1.

The Arabidopsis TGA family of basic leucine zipper transcription factors regulate the expression of pathogenesis-related genes and are required for resistance to disease. Members of the family possess diverse properties in respect to their ability to transactivate and interact with NPR1, the central regulator of systemic acquired resistance in Arabidopsis. Two TGA factors, TGA1 and TGA2, have 83 % amino acid similarity but possess differing properties. TGA1 does not interact with NPR1 but is able to transactivate, while TGA2 interacts with NPR1 but is unable to transactivate. This study uses these two TGA factors to identify amino acids that are responsible for their function. <p>Four cysteines residues within TGA1 were targeted for study by site-directed mutagenesis and the resulting mutants were tested for interaction with NPR1 in yeast. The construct containing a mutation of cysteine 260 (Cys-260) interacted well with NPR1, while those with mutations at Cys-172 or Cys-266 interacted poorly. The Cys-260 mutant also displayed the greatest decrease in transactivation potential in yeast, while mutation of Cys-172 or Cys-266 resulted in smaller decreases. Mutation of Cys-287 had no effect on NPR1 interaction or transactivation. Combining various point mutations in a single protein did not increase NPR1 interaction or transactivation levels, indicating that Cys-260 is crucial for regulating TGA1 properties. Cysteines possess the unique ability of forming reversible disulfide bonds which have been shown to regulate several mammalian cellular processes. The observation that mutation of a single TGA1 cysteine (Cys-260) greatly alters the proteins properties provides a convincing argument that oxidoreduction of this residue is important for its regulation, possibly through the formation of a disulfide bond with either Cys-172 or Cys-266. <p>To test whether other members of the TGA family could be regulated by oxidoreduction, several TGA2 constructs were created that introduced Cys at positions corresponding to those found in TGA1. When tested in yeast none were able to transactivate but continued to interact with NPR1.

The Non-expressor of pathogenesis-related1 gene (NPR1) mediates the induction of pathogenesis-related (PR) gene products, vital for resistance in plants. In this study, the role of two previously isolated Cavendish banana NPR1-like genes (MNPR1A and MNPR1B) has been characterized in protection against Xanthomonas campestris, Hylaperonospora arabidopsidis, Botrytis cinerea and Pseudomonas syringae pathogens. The specific aim was to investigate if sequence differences in both genes are responsible for differential activity against pathogens because in a previous expression study, MNPR1A and not MNPR1B had been more responsive to the banana necrotrophic pathogen Fusarium oxysporum. By challenging Fusarium-tolerant GCTCV-218 and susceptible Grand Naine Cavendish banana plants (which had been used in a previous characterization study) with the hemi-biotrophic Xanthomonas pathogen (a very important economical pathogen of banana), the two MNPR1, PR-1 and PR-3 genes were found to be sequentially expressed. Expression of these genes was more pronounced in the tolerant GCTCV-218 banana cultivar than in the sensitive Grand Naine cultivar. Comparative sequence analysis further showed that these two banana NPR1-like coding sequences had dissimilarities even within conserved functional domains; they grouped closely with other defense-related NPR1-like sequences and harboured defense cis-regulatory elements. Transformation of the coding sequences of both genes under the control of the 35S CaMV promoter/terminator sequences into npr1-2 Arabidopsis mutant complimented the phenotype of this mutant following infection with distinct classes of pathogens (biotrophic Hyaloperonospora, necrotrophic Botrytis and hemi-biotrophic Pseudomonas pathogens). These Infected-MNPR1-expressing plants had higher PR-1 transcript amounts with more reduced pathogen growth compared to non-transgenic npr1-2 Arabidopsis mutant plants. However, the difference in the two banana coding sequences did not translate into a differential pattern of response against the three different classes of pathogens used in this study. Further detailed studies are suggested to investigate the role of the MNPR1 promoter-coding sequences in the differential response to pathogens using a bananapathogen system. This study also addressed the question of whether cystosolic glutathione (GSH) is necessary for NPR1 transcription during systemic acquired resistance. Using Arabidopsis mutants (clt1clt2clt3) defective in cytosolic GSH biosynthesis and following infection with either Pseudomonas or Botrytis, NPR1 and PR-1 transcription was much reduced rendering the mutants more sensitive to pathogens compared to infected-wild-type i>Arabidopsis plants. Results from this study therefore implicate cytosolic glutathione as an essential antioxidant for the establishment of an effective defense response cascade. / Thesis (PhD)--University of Pretoria, 2011. / Plant Science / unrestricted

Plants and pathogenic microorganisms are in constant conflict with each other. Understanding the molecular networks that trigger resistance, along with the molecular networks that pathogens might co-opt to infect susceptible plants, is important for developing the integrated, holistic perspective that is necessary for innovative development of engineered resistance to current and emerging pathogens. The first objective of the dissertation was to increase the understanding of mechanisms by which plants recognize pathogen attack and mount an appropriate defense response. These experiments focused on resistance triggered by the Arabidopsis thaliana R gene, RPP7, which encodes a coiled-coil nucleotide binding-leucine-rich repeat (CC-NB-LRR) protein that activates race-specific resistance to the downy mildew pathogen, Hyaloperonospora parasitica (Hpa). Previously-published genetic epistasis tests have established that RPP7 activates defense responses through a signaling mechanism that does not require accumulation of salicylic acid (SA), or components of the ethylene and jasmonate response pathways. Furthermore, RPP7 is not strongly compromised by mutations in genes associated with defense signal transduction (PAD4, NDR1, NPR1, RAR1). Double mutant combinations of these signal transduction components were analyzed to detect additive or functionally-redundant contributions to RPP7-dependent resistance. Most of the double mutants support an enhanced level of asexual sporulation compared to the single mutant parental lines. Time-course experiments with histochemical stains revealed that these double mutants delay, but do not suppress, the oxidative burst and the hypersensitive response. These results suggest that RPP7 activates multiple signaling pathways, each of which makes incremental contributions to the timing of defense activation. The second objective of the dissertation was to investigate the role that auxin plays in enabling virulent H. parasitica to colonize Arabidopsis. Transcript profiling revealed induction of auxin-associated genes in response to infection of Arabidopsis thaliana by virulent strains of the oömycete pathogen, H. parasitica. Experiments with the DR5 / Ph. D.

No description available.

La vigne (Vitis vinifera) est sensible à de nombreuses maladies, nécessitant l'utilisation massive de produits phytosanitaires. Des méthodes alternatives doivent être développées. Chez la plante modèle Arabidopsis thaliana, les gènes AtNPR1, AtNDR1 et AtEDS1 interviennent dans la voie de signalisation contrôlée par l'acide salicylique (SA) en réponse aux agents pathogènes biotrophes. Nous avons identifié par une approche gène candidat chez V. vinifera, de potentiels orthologues de ces trois gènes (VvNPR1.1, VvNPR1.2, VvNHL1 et VvEDS1).Nos travaux ont montré que les protéines VvNPR1 fusionnées à la GFP sont localisées dans le noyau. De plus, la surexpression transitoire des VvNPR1 stimule l'expression de gènes de défense en système hétérologue et homologue. La surexpression de VvNPR1.1 chez le mutant npr1 restaure les différents phénotypes du mutant, contrairement à la surexpression de VvNPR1.2. VvNPR1.1 est semble être l'orthologue d'AtNPR1. La surexpression de VvNHL 1 chez le mutant ndr1 ne rétablit pas la résistance à Pseudomonas syringae. Cependant, ces plantes sont plus sensibles à Botrytis cinerea, probablement par l'accentuation d'un processus de mort cellulaire. L'expression de VvNHL 1 est réprimée en réponse à B. cinerea, constituant un mécanisme de défense contre les nécrotrophes. VvNHL1 semble donc promouvoir la mort cellulaire. L'expression de VvEDS1 est stimulée en réponse à des agents pathogènes et à un traitement par le SA. L'étude de sa fonction est en cours par surexpression chez des mutants eds1. La compréhension des voies de signalisation des réponses de défense chez la vigne devrait permettre l'obtention de vignes plus résistantes aux agents pathogènes. / Vitis vinifera is susceptible to many pathogens, thus requiring a massive use of phytochemicals. Alternative methods have to be developed. In the madel plant Arabidopsis thaliana, the AtNPR1, AtNDR1 and AtEDS1 genes are components of the salicylic acid (SA) signalling pathway involved in resistance to biotrophic pathogens. Using a candidate gene approach, we have identified putative orthologs of these genes in the grapevine genome (VvNPR1.1, VvNPR1.2, VvNHL 1 and VvEDS1).Our work shows thal VvNPR1-GFP fusion proteins are localized in the nucleus. Moreover, transient overexpression of VvNPR1 genes induces defence gene expression, in bath heterologous and homologous systems. Overexpression of VvNPR1.1 in npr1 mutants restores the different phenotypes of the mutants, while VvNPR1.2 overexpression does not. These results strongly suggest that VvNPR1.1 is the AtNPR1 ortholog. Overexpression of VvNHL 1 in ndr1 mutants does not restore resistance to Pseudomonas syringae. However, resistance to Botrytis cinerea in these plants is weakened, probably because of stimulation of cell death. Furthermore, VvNHL1 repression following B. cinerea infection in V. vinifera could be considered as a defence mechanism against necrotrophic pathogens and suggests thal VvNHL1 is involved in promotion of cell death. VvEDS1 expression is induced by pathogens infection and SA treatment. VvEDS1 functional characterization is in progress using eds1 Arabidopsis mutants overexpressing VvEDS1. Knowledge of defence signalling pathways in V. vinifera will contribute to obtain pathogen resistant grapevines.

La vigne est soumise à de nombreuses maladies impliquant l'utilisation de produits phytosanitaires en grande quantité dont l'utilisation est néfaste pour l'environnement et la santé des utilisateurs. Un enjeu est donc de développer des méthodes alternatives à la lutte chimique. La protéine codée par le gène NPR1 (Nonexpressor of pathogenesis-related gene 1) joue un rôle clef dans la résistance à large spectre chez les plantes. Des éliciteurs tels que l'acide salicylique ou des agents pathogènes influencent l'activation de NPR1 dans le cytoplasme. La translocation de NPRl dans le noyau et son interaction avec des facteurs de transcription TGA induit l'expression des gênes PR (Pathogenesis-related). Nous avons identifié sept homologues potentiels des gènes NPR1 et TGA chez Vitis vinifera (VvNPR1.1, VvNPR1.2, VvTGA1 à 5). L'étude de la diversité de séquences dans les exons de 15 accessions de Vitaceae indique qu'ils sont soumis à une forte pression de sélection purificatrice. De plus, l'analyse in silico des régions promotrices des VvNPR1 montre la présence, d'éléments cis-régulateurs potentiels, en réponse aux stress biotiques et abiotiques ainsi que des motifs de liaison à des facteurs de transcription. Une étude plus poussée des introns montre quelques éléments transposables et un faible polymorphisme dans six accessions de Vitis vinifera. Ces résultats argumentent en faveur d'une pression de sélection forte agissant sur ces gènes. Ceci nous a mené à formuler des hypothèses fonctionnelles et à réaliser une étude d'interaction avec les facteurs de transcription VvTGA1 et VvTGA4 par la technique du double hybride. Ces derniers n'interagissent pas avec VvNPR 1.1. / Numerous diseases affect grapevine, resulting in the use of phytochemicals in large quantities that are harmful for environment and user's health. In the long term, the aim is to develop alternative methods to chemicals. The protein encoded by NPR1 (Nonexpressor of pathogenesis-related gene 1) plays a pivotal role in conferring broad spectrum pathogen resistance in plants. Activation of NPR 1 in the cytoplasm is influenced by elicitors such as salicylic acid or pathogens associated with the accumulation of reactive oxygen species. Translocation of NPR1 into the nucleus and interaction with TGA transcription factors induce the expression of PR (Pathogenesis-related) genes. Using a candidate gene approach, we have identified seven putative homologs to NPR1 and TGA in the grapevine genome (VvNPR1.1, VvNPR1.2, VvTGA1 to 5). The study of sequence diversity in exons of 15 accessions of the Vitaceae family indicates that these exons are subjected to a strong purifying selection pressure. Moreover, in silico analysis in the promoters of VvNPR1 shows putative cis-regulator elements, in answer to biotic and abiotic stresses as well as link patterns to transcription factors. An intron study shows transposable elements and a low polymorphism in six accessions of Vitis vinifera. These results suggest a strong selection pressure on these genes. Functional hypotheses were formulated, and an interaction study with transcription factors VvTGA1 and VvTGA4 was conducted using a method based on yeast two hybrid, showing that they do not interact with VvNPR1.1.

Biotic and abiotic stresses are signifi cant factors limiting the production of food and other supporting materials required to sustain increasing world population. Plant health is directly related to human health and is increasingly becoming signifi cant and demands more attention towards limiting the damages caused by biotic and abiotic stresses. Signifi cant progress has been made towards our understanding of the processes, which mediate both biotic and abiotic stress signaling in plants. Signifi cant role is played by various plant hormones, e.g., salicylic acid (SA) and jasmonic acid (JA) in biotic stress and abscisic acid (ABA) in abiotic stress (Annu Rev Cell Dev Biol 28:489-521, 2012). Other hormones with minor role include the cytokinins (CK), auxins (indole 3 acetic acid. IAA), and the brassinosteroids (BR) (Annu Rev Cell Dev Biol 28:489-521, 2012). Cross talk between these plant hormones is signifi cant and may result in either synergistic or antagonistic effect on stress responses (Annu Rev Cell Dev Biol 28:489-521, 2012). In recent years, extensive research carried out in various laboratories has implicated cross talk between the ABA and the SA in abiotic stress response. This is signifi cant in light of SA being key player in biotic stress responses in plants. This review will discuss the role of SA in biotic and abiotic stress signaling and its cross talk with other hormones in mediating abiotic stress signaling in plants.

Salicylic acid (SA) is a key plant hormone that mediates host responses against microbial pathogens. Identification and characterization of SA-interacting/binding proteins is a topic which has always excited scientists studying microbial defense response in plants. It is likely that discovery of a true receptor for SA may greatly advance understanding of this important signaling pathway. SABP2 with its high affinity for SA was previously considered to be a SA receptor. Despite a great deal work we may still not have true a receptor for SA. It is also entirely possible that there may be more than one receptor for SA. This scenario is more likely given the diverse role of SA in various physiological processes in plants including, modulation of opening and closing of stomatal aperture, flowering, seedling germination, thermotolerance, photosynthesis, and drought tolerance. Recent identification of NPR3, NPR4 and NPR1 as potential SA receptors and α-ketoglutarate dehydrogenase (KGDHE2), several glutathione S transferases (GSTF) such as SA binding proteins have generated more interest in this field. Some of these SA binding proteins may have direct/indirect role in plant processes other than pathogen defense signaling. Development and use of new techniques with higher specificity to identify SA-interacting proteins have shown great promise and have resulted in the identification of several new SA interactors. This review focuses on SA interaction/binding proteins identified so far and their likely role in mediating plant defenses.

Interés del estudio: Profundizar en el conocimiento de algunos de los mecanismos de resistencia que han desarrollado las plantas frente a patógenos, tomando como referencia principal la interacción entre Arabidopsis thaliana y Pseudomonas syringae. Objetivos: 1- Desarrollar y validar un nuevo modelo para el estudio de la percepción del ácido salicílico (SA) basándose en el crecimiento de la planta, no en la inoculación de un patógeno y el posterior análisis de su crecimiento. 2- Aplicar este nuevo modelo para obtener información sobre las señales que empiezan tras la aplicación y/o inducción del SA y que desencadenan la activación de las defensas de la planta. Para ello se realizará un rastreo genético con el que se pretende responder a la pregunta de si el gen NPR1 es el único necesario para la percepción de esta hormona. 3- Clasificar los candidatos obtenidos por grupos de complementación. Mapear aquel grupo más interesante e iniciar su caracterización fenotípica. Elementos de la metodología a destacar: Se han utilizado metodologías tanto comunes a toda la biología molecular de plantas (mutagénesis, secuenciación de DNA, análisis de QTLs, inmunodetección de proteínas, cultivo in vitro de plantas, expresión transitoria de proteínas, doble híbrido en levadura, RT-qPCR, microscopía, análisis de transcriptomas, etc.), como más específicas del campo de la interacción planta-patógeno (cultivo de bacterias, inoculación de bacterias, tratamientos con inductores de resistencia, etc.). Resultados logrados: El análisis de la reducción del peso fresco de la planta provocada por la inducción de resistencia tras la aplicación del BTH (análogo del SA) constituye un modelo biológico válido y eficaz para el estudio de la percepción del SA en Arabidopsis. Además, permite realizar un rastreo genético en busca de mutaciones que afecten a la percepción del SA que ha permitido concluir que el gen NPR1 no es el único implicado en dicho proceso. Por una parte, los cinco parálogos de / Canet Perez, JV. (2012). Análisis genético de la percepción del ácido salicílico en Arabidopsis thaliana. Caracterización de NRB4 [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/16962