The parasitic angiosperm Orobanche aegyptiaca (Pers.) (Egyptian broomrape) is a root holoparasite that causes severe losses in yield and quality of many crops. Control of Orobanche is extremely challenging, in part because the parasite is hidden underground for most of its life cycle. However, the dependence of the parasite on the host suggests that broomrape-resistant hosts could be an ideal control method. Genetic engineering strategies may facilitate realization of this goal, but require an understanding of host defense responses to parasitism. Previous studies with tobacco indicated that broomrape parasitism induces host genes associated with jasmonic acid (JA)-mediated defenses such as wound responses and localized production of phenylpropanoid and isoprenoid phytoalexins. However, the gene for the pathogenesis-related (PR) protein, PR-1a, was not induced by parasitism in tobacco. Expression of PR-1a is correlated with the salicylic acid (SA)-mediated defense pathway that leads to systemic acquired resistance (SAR). The objective of this research was to extend the characterization of PR gene expression in order to define the scope of host defense response. Analyses of gene expression using RNA hybridization and RT-PCR in broomrape-parasitized Arabidopsis thaliana roots indicated that PR-1, PR-2, PR-5, as well as the JA-associated PDF1.2, were slightly induced by parasitism. Expression of PR-1, PR-5, and PDF1.2 in parasitized roots was not detectable by RNA hybridization analysis, but was demonstrated by RT-PCR. Interestingly, shoots of the parasitized plants showed greater PR gene expression levels than roots, indicating that O. aegyptiaca induced a response in the host that was systemic and amplified in shoots. Microarray analysis of parasitized Arabidopsis roots demonstrated a broad range of host gene expression changes including both defense- and non-defense-related genes. Genes induced were consistent with O. aegyptiaca preferentially stimulating JA-mediated responses. The failure of O. aegyptiaca to elicit SA-mediated defenses in host roots suggested that exogenous induction of this signaling pathway could enhance host resistance to parasitism. Treatment of O. aegyptiaca-inoculated tobacco with BTH, a SA analog that activates SAR, caused a 49% reduction in O. aegyptiaca numbers. Analysis of PR-1a using RNA hybridizations and protein immunoblots in treated plants showed the expected induction in shoots, but not in roots, confirming the organ-specific differences in defense response observed in Arabidopsis. Experiments using a strategy to engineer the hypersensitive response via the gene-for-gene interaction confirmed previous findings that parasite-specific activation of an R/Avr interaction in tobacco reduced parasitism by approximately 50%. This research suggests that approaches to stimulate SAR in susceptible host plants may be useful for reducing Orobanche parasitism / Master of Science

The fungal pathogen S. sclerotiorum (Sclerotinia sclerotiorum) impacts production and yield in one of Canada’s number one crops, canola (Brassica napus). Unfortunately, few cultivars show any tolerance to this devastating fungal pathogen. Thus, understanding how the plant responds to this aggressive fungus at the cellular level will facilitate the identification of genes and gene products responsible for improved plant performance. While our understanding of the host pathogen interaction is becoming clearer, there is remarkably little information available for Sclerotinia, especially its pathogenicity in canola. Moreover, we know nothing about how this interaction is specified at the cellular, physiological or molecular level directly at the site of infection in mature leaves following petal inoculation. Thus, we compared differences in plant structure, antioxidant response, and genes involved in the salicylic acid, jasmonic acid and ethylene defense pathways in a susceptible cultivar, Westar, and a previously described tolerant cultivar, Zhongyou821 (ZY821). Our data showed that at the cellular level, ZY821 was able to suppress the Sclerotinia penetration. The ascorbate-glutathione pathway and resistant signaling pathways were all associated with the canola defense response to S. sclerotiorum, while stronger antioxidant and signaling pathways responses were observed in ZY821 leaves at the site of infection. Also, transcriptional regulators not previously associated with plant defense in the Arabidopsis- S. sclerotiorum pathosystem were identified through bioinformatics approaches. By comparing plant susceptibility to S. sclerotiorum between Arabidopsis wild type and seven loss-of-function mutants, I found transcription factor JAM2 might be involved in plant tolerance to S. sclerotiorum. / October 2014

Verticillium wilt, caused by the soil-borne Verticillium dahliae Klebahn is a serious problem in the production of sunflower worldwide. To date, information on sunflower resistance to Verticillium spp. is very scarce, although it is critical for an effective management of this pathogen. In this study, two highly aggressive (Vd1396-9 and Vd1398-21) and two weakly aggressive V. dahliae isolates (Vs06-07 and Vs06-14) were used to inoculate moderately resistant (IS6111) and susceptible (IS8048) sunflower hybrids. VdNEP (V. dahliae necrosis and ethylene-inducing protein), an elicitor from V. dahliae, was also used to infiltrate sunflower plants. Our results indicate that VdNEP has a dual role in the interaction between sunflower and V. dahliae. VdNEP acted not only as a pathogenicity factor on sunflower by inducing wilting symptoms such as chlorosis, necrosis and vascular discoloration, but also as an elicitor triggering defense responses of the host. VdNEP induced the hypersensitive cell death in Nicotiana benthamiana leaves and sunflower cotyledons. Moreover, VdNEP activated the production of reactive oxygen species and the accumulation of fluorescent compounds in sunflower leaves. Pathogenesis-related genes (Ha-PR-3, and Ha-PR-5), two defensin genes (Ha-PDF and Ha-CUA1) and genes encoding Ha-ACO, Ha-CHOX, Ha-GST and Ha-SCO were up-regulated by VdNEP, suggesting that multiple signaling pathways are involved in this interaction. Two SA-related genes (Ha-PAL and Ha-NML1) were slightly suppressed after infiltration with VdNEP, suggesting a possible involvement of VdNEP in affecting sunflower defenses.

Verticillium wilt, caused by the soil-borne Verticillium dahliae Klebahn is a serious problem in the production of sunflower worldwide. To date, information on sunflower resistance to Verticillium spp. is very scarce, although it is critical for an effective management of this pathogen. In this study, two highly aggressive (Vd1396-9 and Vd1398-21) and two weakly aggressive V. dahliae isolates (Vs06-07 and Vs06-14) were used to inoculate moderately resistant (IS6111) and susceptible (IS8048) sunflower hybrids. VdNEP (V. dahliae necrosis and ethylene-inducing protein), an elicitor from V. dahliae, was also used to infiltrate sunflower plants. Our results indicate that VdNEP has a dual role in the interaction between sunflower and V. dahliae. VdNEP acted not only as a pathogenicity factor on sunflower by inducing wilting symptoms such as chlorosis, necrosis and vascular discoloration, but also as an elicitor triggering defense responses of the host. VdNEP induced the hypersensitive cell death in Nicotiana benthamiana leaves and sunflower cotyledons. Moreover, VdNEP activated the production of reactive oxygen species and the accumulation of fluorescent compounds in sunflower leaves. Pathogenesis-related genes (Ha-PR-3, and Ha-PR-5), two defensin genes (Ha-PDF and Ha-CUA1) and genes encoding Ha-ACO, Ha-CHOX, Ha-GST and Ha-SCO were up-regulated by VdNEP, suggesting that multiple signaling pathways are involved in this interaction. Two SA-related genes (Ha-PAL and Ha-NML1) were slightly suppressed after infiltration with VdNEP, suggesting a possible involvement of VdNEP in affecting sunflower defenses.

The necrotrophic fungus Botrytis cinerea (B. cinerea) is one of the most aggressive and widespread pathogenic fungi, causing the gray mold disease in over 200 different plants species and severely impacting the quality of harvested fruits. Due to B. cinerea resistance to common synthetic fungicides and their ecological impact, there is a large demand for novel control approaches, such as bio-fungicides. The family of carotenoid-cleavage products, i.e. apocarotenoids, includes important compounds, such as hormones, signaling molecules and growth regulators. Previous studies indicated that the apocarotenoid B-ionone inhibits the growth of some fungal species. In this thesis, we unravel a novel role for B-ionone as a plant regulatory metabolite increasing the resistance against B. cinerea in several plant models. We achieved this by combining phenotypic, transcriptomic, and metabolomic analysis. We show that pretreatment of Arabidopsis plants with B-ionone significantly alleviated the symptoms of B. cinerea infection, modulated hormone homeostasis and affected the interactions between jasmonic acid (JA)/ethylene (ET) and abscisic acid (ABA) hormone signaling pathways β-ionone treatment stimulated JA/ET signaling pathways and repressed the synthesis of ABA upon B. cinerea infection, which reduced the susceptibility of Arabidopsis plants to B. cinerea. To get an overview on the effect of β-ionone on plants at transcript level, we performed an RNA-seq experiment that supported our hypothesis that B-ionone primes and enhances the Arabidopsis immune response to B. cinerea infection and raveled the effect of this volatile on the expression of several transcription factors involved in Arabidopsis immune response to B. cinerea and on transcripts related to cell wall biosynthesis, Map Kinase 3 signaling and hypoxia tolerance. Further experiments performed with transgenic tobacco and tomato plants confirmed the positive role of β-ionone in reducing the symptoms of B. cinerea in green tissues as well as in tomato fruits. The discovery of β-ionone as a new apocarotenoid signaling molecule that regulates plant hormone homeostasis opens up new possibility to control B. cinerea infection and to establish this natural volatile as an environmentally friendly and safe bio-fungicide.

Un des principaux acteurs impliqués dans la régulation du statut redox intracellulaire, permettant aux plantes de s’adapter aux contraintes environnementales, est une famille multigénique de petites (12-14 kDa) oxydoréductases ubiquistes appelées thiorédoxines (TRX). Le génome d’Arabidopsis code une vingtaine de TRX canoniques dont neuf sont plastidiales (TRX f, m, x et y). Très étudiées dans notre laboratoire par des approches biochimiques, les TRX de types f et m apparaissent réguler majoritairement l’activité d’enzymes impliquées dans le métabolisme primaire tandis que les types x et y servir principalement de substrats réducteurs d’enzymes antioxydantes. Plus récemment, une dixième TRX, proposée plastidiale et nommée TRX z, a été identifiée. Au cours de ma thèse, j’ai caractérisé cette nouvelle TRX chloroplastique montrant des propriétés physico-chimiques inhabituelles, la rendant unique. En effet, la TRX z semble interagir, via des interactions électrostatiques, avec des protéines pour former des complexes de masses moléculaires élevées, potentiellement liés aux acides nucléiques. De plus, la TRX z, dont l’expression est induite à la lumière, principalement dans les tissus photosynthétiques, est la première TRX chloroplastique qui n’est pas réduite par le système FTR à la lumière mais qui peut être réduite par les autres TRX plastidiales, suggérant une interconnexion entre ces différentes TRX. D’autre part, une recherche exhaustive de cibles de la TRX z, par deux approches spécifiques et complémentaires (protéomique et double hybride), ont révélé 90 cibles putatives de la TRX z. La plupart de ces cibles, jamais identifiées comme cibles des TRX, sont impliquées dans la réponse de défense des plantes mise en place lors de stress biotiques. Ces résultats suggèrent que la TRX z pourrait être un élément clé dans la mise en place de ces réponses. L’analyse fonctionnelle préliminaire de la TRX z au cours de la réponse immune innée conforte cette hypothèse. L’ensemble de ces résultats indique que la TRX z pourrait jouer le rôle d’une protéine senseur de l’état d’oxydoréduction de la cellule. / One of the main actors involved in regulation of the cellular redox state, which allow plant adaptation to stress environmental conditions, is a multigenic family of small (12-14 kDa) ubiquitous oxidoreductases named thioredoxins (TRX). Arabidopsis encodes around twenty canonical TRX, including nine plastidial isoforms (TRX f, m, x and y). Extensively studied in our laboratory by biochemical approaches, TRX f and m was found to mainly redox regulate the activity of enzymes involved in the primary metabolism whereas TRX x and y serve as reducing substrates for antioxidant enzymes. More recently, a tenth TRX, predicted plastidial and named TRX z, was identified. During my PhD, I have characterized this new plastidial TRX showing unusual physicochemical properties, making it unique. Indeed, TRX z seems to interact, via electrostatic bonds, with proteins to form high molecular weight complexes, potentially linked to nucleic acids. Moreover, TRX z, which is expressed in green tissues in the light, is the first plastidial TRX which is not reduced by the FTR system but which can be reduced by other plastidial TRX, suggesting an interconnection between these TRX. Furthermore, a large scale inventory of TRX z targets, by two specific and complementary approaches (proteomic and yeast two hybrid), revealed 90 putative TRX z targets. Most of these, which have never been identified as TRX targets before, are implicated in plant defense response to biotic stresses. These results suggest that TRX z might be a key player in these responses. Preliminary functional analysis of TRX z during immune innate response reinforces this hypothesis. Altogether, these results indicate that TRX z appears as an important sensor of the redox status of the cell.