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.

Plants are sessile organisms adapted to cope with dynamic changes in their environment. Abiotic stresses, such as heat, drought, or nutrient deficiency must be overcome simultaneously with biotic threats such as pathogens and herbivores. Oomycete pathogens represent a significant threat to global food production and natural ecosystems. Novel modes of oomycete disease control could increase crop yield and reduce pesticide application. Overlaps between the plant response to iron deficiency and pathogens have been documented, but the impact of simultaneous imposition of both stresses on the plant have not been studied. Additionally, nothing is known about the impact of iron deficiency on oomycete infection, or mechanisms of oomycete iron uptake. We adapted a hydroponic system to simultaneously impose iron deficiency and monitor pathogen infection. The oomycete pathogens Hyaloperonospora arabidopsidis, and Phytophthora capsici grew less well on iron-deficient Arabidopsis thaliana, at least in part because of observed activation of immunity due to iron stress. We screened A. thaliana T-DNA insertion mutants defective in iron metabolism and transport and identified potential mechanisms of H. arabidopsidis iron acquisition. We conducted RNA sequencing to understand how A. thaliana responds to iron deficiency and root infection of P. capsici. 323 genes were differentially upregulated in iron-starved plants over three days, irrespective of pathogen infection, representing a core iron deficiency response. This group of core genes included the primary A. thaliana iron uptake pathway and genes for coumarin biosynthesis. Salicylic acid responsive genes were observed in both treatments consistent with this defense hormone's previously identified role in iron deficiency. Genes related to glucosinolate production – shown to be important in defense against P. capsici – were down regulated during infection, potentially due to the activity of virulence effectors. Our work demonstrates crosstalk between the iron deficiency response and plant immunity, and that iron acquisition remains important to the plant even after pathogen invasion. These new insights provide a first step in developing novel resistance strategies to control oomycetes in agronomically important crops. / Doctor of Philosophy / Oomycetes can cause diseases in plants resulting in loss of crops and requiring application of chemical pesticides. Better understanding of how oomycetes interact with plants will lead to new strategies to control them and more efficient agriculture. In this study, we investigated the role of iron in plant-oomycete interactions, to see what this important metal nutrient might be doing to help or hurt the plants response to infection. We developed a growth system to limit iron to the plant and simultaneously observe oomycete infection. We studied the leaf pathogen Hyaloperonospora arabidopsidis or downy mildew, and Phytophthora capsici, a root pathogen that infects many types of vegetable crops. In rice, iron restriction hurt the plant's ability to fight off disease, but we observed the opposite: iron limitation caused the plant to be more resistant to both oomycete pathogens. Microscopic observation revealed that the plants ability to fight off downy mildew was not compromised by iron deficiency. Our results suggest that iron limitation triggers an immune response in the plant, which limits pathogen growth. We performed RNA sequencing on iron-deficient roots also infected with the root pathogen. This allowed us to observe how the plant responded to both stresses. The plant balances the response to iron deficiency and infection. Again, we found that iron deficiency triggers immune activation, and observed that iron-deficient plants are more resistant to infection.

Effector proteins are exported to the interior of host cells by numerous plant pathogens. Effector proteins have been well characterized in bacteria. However, the mechanisms through which these effectors promote virulence are largely unknown. Bioinformatic analysis of genome sequences from oomycete pathogens Phytophthora sojae, P. ramorum, P. infestans and Hyaloperonospora arabidopsidis (Hpa) have led to the identification of a large number of candidate effector genes. These effector genes have characteristic motifs (signal peptide, RxLR and dEER) that target the effectors into plant cells. Although these effector genes are very diverse, certain genes are conserved between P. sojae and H. arabidopsidis, suggesting that they play important roles in pathogenicity. The goal of my first project was to characterize a pair of conserved effector candidates from Hpa and P. sojae. We hypothesized that these effectors have important conserved roles with regard to infection. We found that the Hpa effector was expressed early during the course of infection of Arabidopsis and triggered an ecotype-specific defense response in Arabidopsis, suggesting that it was recognized by host surveillance proteins. Both the effectors from Hpa and P. sojae respectively could suppress immunity triggered by pathogen associated molecular patterns (PTI) and by effectors (ETI) in planta. They also enhanced bacterial virulence in Arabidopsis when delivered by the Type III secretion system. Similar results were seen with experiments with transgenic Arabidopsis expressing the effectors. My second project showed that a different Hpa effector protein, HaRxL10, targets the Jasmonate-Zim Domain (JAZ) proteins that repressed responses to the phytohormone jasmonic acid (JA). This manipulation activates a regulatory cascade that reduces accumulation of a second phytohormone, salicylic acid (SA) and thereby attenuates immunity. This virulence mechanism is functionally equivalent to but mechanistically distinct from activation of JA-SA crosstalk by the bacterial JA mimic coronatine. These results reveal a new mechanism underpinning oomycete virulence and demonstrate that the JA-SA crosstalk is an Achilles\' heel that is manipulated by unrelated pathogens through distinct mechanisms. / Ph. D.

: Les plantes ont développé au cours de leur évolution un système d’immunité innée constitué de barrières préformées et de réponses de défense induites contre les agents pathogènes. Ce travail s’inscrit dans l’étude des résistances induites chez Arabidopsis thaliana soit naturellement contre les agents pathogènes racinaires Fusarium oxysporum spp. (Fo), ou après application de phosphite (Phi), contre le parasite foliaire Hyaloperonospora arabidopsidis (Hpa). Dans la première partie du travail, les rôles des métabolites secondaires (MS) et des formes réactives de l’oxygène (ROS) dans les résistances racinaires basale et non-hôte, respectivement aux formes spéciales conglutinans (Foco) et melonis (Fom) de Fo, ont été analysés. Nous avons démontré la participation de la camalexine, une phytoalexine indolique, dans la résistance basale d’Arabidopsis à Foco. En revanche, la scopolétine, une phytoalexine phénolique, et les ROS jouent des rôles essentiels dans la résistance non-hôte à Fom. Ces données soulignent le rôle clé des MS et des ROS dans les résistances hôte et non-hôte d’Arabidopsis. Dans une deuxième partie de ce travail, le mode d’action du Phi, un oxyanion de l’acide phosphoreux (H3PO3) protégeant Arabidopsis contre l’isolat Noco2 de Hpa, a été étudié. L’effet de faibles doses de Phi est aboli chez des mutants d’Arabidopsis affectés dans la voie de transduction du signal acide salicylique (SA) indiquant que l’induction de résistance à Hpa est médiée par des mécanismes dépendants du SA. Le Phi potentialise les réponses de défense contre Hpa Noco2 via EDS1-PAD4, deux composants essentiels de la résistance basale, NPR1 et la protéine de défense PR1. L’expression de la MAP kinase MPK4, un régulateur négatif de la résistance à Hpa, est diminuée par le Phi, lors de l’inoculation par Hpa Noco2. Nos résultats démontrent que la potentialisation des réponses de défense par le Phi est associée à la régulation négative de MPK4. / Plants have developed during their evolution an innate immunity system consisting of preformed barriers and induced defence responses against pathogens. This work studies resistances in Arabidopsis thaliana induced either naturally against the root pathogen Fusarium oxysporum spp. (Fo), or after application of phosphite (Phi) against the leaf pathogen Hyaloperonospora arabidopsidis (Hpa). In a first part, roles of secondary metabolites (SM) and reactive oxygen species (ROS) in basal and non-host resistances of roots to the special forms conglutinans (Foco) and melonis (Fom) of Fo, respectively, were analyzed. We demonstrated the involvement of the indolic phytoalexin camalexin, in basal resistance of Arabidopsis to Foco. In contrast, the phenolic phytoalexin, scopoletin, and ROS play essential roles in non-host resistance to Fom. These data underscore the key role of MS and ROS in basal and non-host resistances of Arabidopsis. In a second part, the mode of action of Phi, an oxyanion of phosphorous acid (H3PO3) protecting Arabidopsis against the Hpa isolate Noco2 was studied. Effect of low doses of Phi is abolished in Arabidopsis mutants affected in salicylic acid (SA) signalling, indicating that induced resistance to Hpa is mediated by SA-dependent mechanisms. Phi primes defence responses against Hpa Noco2 via EDS1-PAD4, two essential components of basal resistance, as well as NPR1 and PR1. Expression of the MAP kinase MPK4, a negative regulator of resistance to Hpa, is decreased by Phi after inoculation with Hpa Noco2. Our results demonstrate that priming of defence responses by Phi is associated with down-regulation of MPK4.

Les plantes présentent une forme d’immunité innée face à des agents potentiellement pathogènes qui se traduit par l’induction de réponses de défense. Les réponses immunes des plantes sont induites après détection de motifs moléculaires associés à des pathogènes ou à des micro-organismes par des récepteurs reconnaissant spécifiquement ces motifs et/ou des molécules dérivées des agents pathogènes ou de la plante, appelés éliciteurs de réaction de défense. La cryptogéine (Cry) et les oligogalacturonates (OGs) sont des éliciteurs établis de réactions de défense et leur reconnaissance induit une signalisation Ca2+-dépendante : un influx calcique et une variation de la concentration cytosolique en Ca2+ libre ([Ca]cyt) sont des événements précoces induisant une voie de signalisation de défense. Nous avons démontré que chez le tabac, les éliciteurs induisent une signalisation calcique dans les mitochondries et les chloroplastes. Des études pharmacologiques indiquent que des canaux IP3-dépendants régulent la signalisation calcique induite par la Cry dans les mitochondries et les chloroplastes. La respiration mitochondriale et les mécanismes de dissipation de l’énergie dans les chloroplastes sont régulés en partie par la [Ca2+] dans ces organites. De plus, nous montrons par des approches pharmacologiques et génétiques, que des homologues aux récepteurs du glutamate (GLRs) participent à la signalisation calcique induite par les OGs dans Arabidopsis. Les GLRs contrôlent en partie la production d’oxyde nitrique (NO) et d’espèces réactives de l’oxygène (ROS), ainsi que l’expression de gènes de défense. Par ailleurs, les plantes traitées par des antagonistes des GLRs, présentent une moindre résistance au pathogène fongique nécrotrophique, Botrytis cinerea et à l’oomycète biotrophique, Hyaloperonospora arabidopsidis. L’analyse de mutants Atglr révèle l’importante contribution de AtGLR3.3 dans la résistance envers H. arabidopsidis. De plus, de frappantes similarités dans l’expression de gènes sont observées après traitement par les OGs ou après infection par H. arabidopsidis. Enfin, une analyse transcriptomique montre qu’environ 60 % des gènes modulés par les OGs ont une expression qui dépend de GLRs. Ces gènes dépendants de GLRs appartiennent à diverses familles fonctionnelles dont celle répondant aux stress biotiques. En conclusion, ces études montrent 1) que les mitochondries et les chloroplastes présentent aussi une signalisation calcique induite par des éliciteurs de réaction de défense chez le tabac et 2) l’implication de GLRs dans la signalisation calcique induite par des éliciteurs ou des agents pathogènes et la résistance envers des agents pathogènes chez Arabidopsis / Plants do not display an adaptive immune system but express an efficient innate immune system defending them by inducing sophisticated multilevel defense responses against different potential pathogens. Indeed, plant immune responses are triggered upon the detection of many common pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs) through specific pattern-recognition receptors (PRRs) and/or pathogen- or plant-derived signal molecules called elicitors. Cryptogein (Cry) and oligogalacturonides (OGs) are well known elicitors of defense reactions and their recognition induce a Ca2+-dependent signaling pathway: Ca2+ influx and subsequent free cytosolic [Ca2+] ([Ca2+]cyt) variations are earliest steps to trigger downstream plant defense signaling. Here we have demonstrated that elicitor-induced Ca2+ signaling in tobacco also takes place in mitochondria and chloroplasts. Pharmacological studies indicated that IP3-channels play an important role in the regulation of Ca2+ signaling in mitochondria and chloroplasts. Mitochondrial respiration and energy dissipation mechanisms in chloroplasts are partly controlled by [Ca2+] in these organelles. Moreover, using pharmacological and genetic approaches, our data demonstrated that glutamate receptors homologs (GLRs) participate in OGs-mediated Ca2+ signaling in Arabidopsis. GLRs partly control OGs-induced nitric oxide (NO) production, reactive oxygen species (ROS) production and expression of defense-related genes. Importantly, plants treated with GLRs antagonists exhibited compromised resistance to necrotrophic fungal pathogen, Botrytis cinerea and biotrophic oomycete, Hyaloperonospora arabidopsidis. Analysis of Atglr single mutants revealed the important contribution of AtGLR3.3 in resistance against H. arabidopsidis. Moreover, striking similarities in gene expression levels were observed after OGs elicitation/H. arabidopsidis infection. Finally, transcriptomic analysis demonstrated that about 60 % of the total OGs-modulated genes modified their expression in GLRs-dependent manner. These GLRs-dependent genes belong to different functional categories including the category “responses to biotic stresses”. Taken together, these data provide strong evidences of 1) elicitor-induced Ca2+ signaling in mitochondria and chloroplasts in tobacco and 2) the regulation of elicitor/pathogen mediated plant defense signaling pathways through GLRs in Arabidopsis thaliana

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Plants do not display an adaptive immune system but express an efficient innate immune system defending them by inducing sophisticated multilevel defense responses against different potential pathogens. Indeed, plant immune responses are triggered upon the detection of many common pathogen- or microbe-associated molecular patterns (PAMPs/MAMPs) through specific pattern-recognition receptors (PRRs) and/or pathogen- or plant-derived signal molecules called elicitors. Cryptogein (Cry) and oligogalacturonides (OGs) are well known elicitors of defense reactions and their recognition induce a Ca2+-dependent signaling pathway: Ca2+ influx and subsequent free cytosolic [Ca2+] ([Ca2+]cyt) variations are earliest steps to trigger downstream plant defense signaling. Here we have demonstrated that elicitor-induced Ca2+ signaling in tobacco also takes place in mitochondria and chloroplasts. Pharmacological studies indicated that IP3-channels play an important role in the regulation of Ca2+ signaling in mitochondria and chloroplasts. Mitochondrial respiration and energy dissipation mechanisms in chloroplasts are partly controlled by [Ca2+] in these organelles. Moreover, using pharmacological and genetic approaches, our data demonstrated that glutamate receptors homologs (GLRs) participate in OGs-mediated Ca2+ signaling in Arabidopsis. GLRs partly control OGs-induced nitric oxide (NO) production, reactive oxygen species (ROS) production and expression of defense-related genes. Importantly, plants treated with GLRs antagonists exhibited compromised resistance to necrotrophic fungal pathogen, Botrytis cinerea and biotrophic oomycete, Hyaloperonospora arabidopsidis. Analysis of Atglr single mutants revealed the important contribution of AtGLR3.3 in resistance against H. arabidopsidis. Moreover, striking similarities in gene expression levels were observed after OGs elicitation/H. arabidopsidis infection. Finally, transcriptomic analysis demonstrated that about 60 % of the total OGs-modulated genes modified their expression in GLRs-dependent manner. These GLRs-dependent genes belong to different functional categories including the category "responses to biotic stresses". Taken together, these data provide strong evidences of 1) elicitor-induced Ca2+ signaling in mitochondria and chloroplasts in tobacco and 2) the regulation of elicitor/pathogen mediated plant defense signaling pathways through GLRs in Arabidopsis thaliana