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 understanding of plant defense response in plant-pathogen interaction provides useful information to combat the disease. Soybean cyst nematode (SCN), Heterodera glycines, and charcoal rot causing fungus, Macrophomina phaseolina, are major pathogens of soybean, Glycine max, and infestation with SCN has found to aggravate the fungal disease severity. The gene expression analysis in resistant and susceptible interactions between H. glycines and G. max has identified some candidate resistance genes and signaling pathways but they are yet to be fully characterized. This dissertation aims to characterize one such gene, Conserved Oligomeric Golgi (COG) complex. The COG complex is important in structural and functional integrity of Golgi complex among eukaryotes, but very little is understood about its role in plants. This study demonstrated a defensive role of the COG complex in G. max against H. glycines. The transgenic plants for overexpression of COG genes, which were originally susceptible to the pathogen, showed reduced parasitism. In contrast, the RNA interference (RNAi) in originally resistant soybean lines showed a marked increase in parasitism. Further, these COG genes were found to be inducible by harpin elicitor molecules. In another study, the already proven resistance genes (NDR1, NPR1, EDS1 and TGA2) against H. glycines were investigated against the fungal pathogen. The transgenic plants for overexpression of these genes showed reduced disease severity, while the RNAi resulted increased severity compared to control lines. In addition, the H. glycines parasitism study and the candidate gene expression analysis in M. Phaseolina susceptible and moderately resistant G. max indicate that there are some cross communications between the defense processes of G. max to H. glycines and M. phaseolina.

Cuscuta campestris is an obligate parasitic plant that lacks expanded leaves and roots and requires a host to complete its lifecycle. Parasite-host connections occur via an haustorium, a unique organ that acts as a bridge for the exchange of water, nutrients, macromolecules like mRNA, microRNA, and proteins, and microorganisms. Studies of Cuscuta spp. are challenging due to its dependence on the host and other host influences on the parasite. Recent research has shown intriguing aspects of Cuscuta biology like exchange genetic material with its hosts and loss of genes involved in processes such as high photosynthetic rates and defense. We developed new tools and methodologies that allow us to explore C. campestris biology in an unprecedent way. Foremost of these is an axenic method to grow C. campestris on an Artificial Host System (AHS). The AHS allows C. campestris to display its entire life cycle in vitro, including seed production. Using the AHS, we studied haustorial function, determining the role of nutrients and phytohormones on parasite haustorium development and growth, and found genes involved in haustorial function. The AHS allowed us to demonstrate the positive effect of light on C. campestris growth in the absence of a photosynthetic host and to investigate carotenoid- and ABA- related processes in the haustorial regions. We also wanted to understand how C. campestris defenses work independently from a plant host, so we studied the parasite responses to the bacterial epitope flg22 and the bacteria Peudomonas syringe. Our findings indicate that C. campestris is able to sense flg22, but its response differs from those observed in other non-parasite plants. Transcriptomic analysis revealed up-regulation of genes related to biotic and abiotic stresses, and downregulation of genes related to cuticle development. Our study contributes to understanding the C. campestris immune response in the absence of a host plant. Taken together, this research contributes novel methodologies that enable insights into C. campestris biology without the interference of a plant host on the parasite. / Doctor of Philosophy / Field dodder (Cuscuta campestris) is a parasitic plant that lacks leaves and roots and attacks a wide range of plants, such as tomato and beets. Dodders are not able to carry out full photosynthesis and thus are incapable of producing enough food or obtaining water to survive on their own, so they parasitize other plants. Dodders have developed specialized structures called haustoria that allow them to take resources directly from their hosts. Studying dodder is challenging due to the dependence of the parasite on its host, such that effects of one plant on the other are hard to disentangle. We developed new tools and methodologies that allow us to explore the biology of dodder in unprecedent ways. We developed an Artificial Host System (AHS) that allows the growth and study of dodder without involving a living host plant. Thanks to this new tool, we were able to improve understanding of the function of the haustorium, discover nutrients and growth factors that are indispensable for dodder development, and prove that dodder growth benefits from light. Using the AHS, we compared haustorial regions and shoot tips of dodder to identify genes specific to haustorial function. Additionally, we studied the responses of dodder to bacteria to understand how it reacts to microbial colonization. Our studies contribute with the development of novel methodologies that allow unprecedent discoveries into the biology of dodder. We expect that this work will promote the study of parasitic plant biology.

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.

The infection of plants by Heterodera glycines, commonly known as soybean cyst nematode (SCN), is a serious agricultural problem of worldwide extent. Meanwhile, it provides an excellent experimental model to study basic aspects of how cells function, in particular, during biotic challenge. Heterodera glycines challenges plant cells by initiating, developing and sustaining an interaction that results in the formation of a nurse cell from which the nematode derives nourishment. The presented experiments examine (1) how a cell can be de-differentiated and reprogrammed to perform a much different biological role and (2) how a cell’s immune responses can be engaged or suppressed to accomplish that goal. The observation of alpha soluble N-ethylmaleimide-sensitive factor attachment protein (alpha-SNAP) expression, its location within the rhg1 locus and known involvement in the vesicular transport machinery relating to defense made it a strong candidate for further functional analysis. Functional studies demonstrated that overexpression of alpha-SNAP in the susceptible G. max[Williams 82/PI 518671] genotype that lacks its expression results in the partial suppression of H. glycines infection. This indicated that the vesicles could be delivering cargo to the site of infection to engage a defense response. High levels of expression of a cell wall modifying gene called xyloglucan endotransglycosylase also occur during defense. XTHs associate with vesicles, act in the apoplast outside of the cell, and have a well-known function in cell wall restructuring. These observations indicated that alterations in the cell wall composition of nurse cells could be important for the successful defense response. Overexpression of a G. max xyloglucan endotransglycosylase (Gm-XTH) in the susceptible G. max[Williams 82/PI 518671] genotype resulted in a significant negative effect on H. glycines as well as R. reniformis parasitism. The results, including preliminary experiments on components of the vesicle transport system, identify a potent mechanism employed by plants to defend themselves from two types of plant-parasitic nematodes.

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.

A cana-de-açúcar é um dos cultivos mais importantes no Brasil. Estima-se que uma parte do nitrogênio total de algumas variedades de cana-de-açúcar seja obtida pela interação com microrganismos endofíticos diazotróficos. Na maior parte de sua vida, os endofíticos habitam o interior de plantas sem causar nenhum sintoma aparente de doença. O estudo de interação microrganismos/plantas foi realizado utilizando-se co-culturas entre bactérias endofíticas diazotróficas e calos de cana-de-açúcar. Em presença do calo: Enterobacter sp. ICB113, ICB117 e ICB481 foram estimuladas e mantidas em elevadas populações; Klebsiella sp. ICB375 e Pseudomonas sp. ICB383 tiveram seu número reduzido e mantiveram-se em números estáveis; e Pantoea sp. ICB409 foi totalmente eliminada, demonstrando, assim, que a planta exerce um controle sobre os microrganismos. O modelo calo de cana-de-açúcar/bactéria endofítica diazotrófica foi capaz de evidenciar diferentes interações entre vegetal e bactéria, assim como, diferentes níveis de resposta de defesa da célula vegetal. / The sugarcane is one of the most important crops in Brazil. It is estimated that a portion of the total nitrogen of some varieties of sugarcane is obtained by interaction with diazotrophic endophytic microorganisms. In most of his life, the endophytes inhabit the interior of plants without causing any obvious signs of disease. The interaction study microorganisms/plants was performed using co-cultures among endophytic diazotrophic bacteria and sugarcane calluses. In the presence of callus : Enterobacter sp . ICB113 , ICB117 and ICB481 were stimulated and maintained in large populations ; Klebsiella sp . ICB375 and Pseudomonas sp. ICB383 had their numbers reduced and remained in stable numbers, and Pantoea sp. ICB409 was completely eliminated, thus demonstrating that the plant has a control over microorganisms. The model sugarcane callus/endophytic diazotrophic bacteria was able to show different interactions between plants and bacteria , as well as different levels of defense response of the plant cell.