Utilizing resistant cultivars is a main control strategy for root-knot nematodes in flue-cured tobacco (Nicotiana tabacum L.). Most commercial cultivars possess the Rk1 gene, providing resistance to races 1 and 3 of Meloidogyne incognita and race 1 of M. arenaria. This initiated a shift in root-knot populations to other species and races, creating a need for resistance to those populations. Numerous cultivars possess a second resistance gene, Rk2. Greenhouse experiments investigated whether possessing both Rk1 and Rk2 increases resistance to a variant of M. incognita race 3 compared to either gene alone, and if high soil temperatures impact their efficacy. Root galling, numbers of egg masses and eggs, and the reproductive index were compared from roots of Coker 371-Gold (susceptible), NC 95 and SC 72 (Rk1Rk1), T-15-1-1 (Rk2Rk2), and STNCB-2-28 and NOD 8 (Rk1Rk1 and Rk2Rk2). The same data were analyzed from plants in open-top root zone cabinet growth chambers set to 25ºC, 30ºC, and 35ºC to examine if resistance is temperature sensitive. Despite variability, Rk1Rk2 entries conferred greater resistance than entries with Rk1 or Rk2 alone. Entries with Rk1 alone reduced galling and reproduction compared to the susceptible control, whereas T-15-1-1 (Rk2) did not, but often suppressed reproduction. An apparent reduction in nematode reproduction was observed at 25ºC and 30ºC on entries possessing Rk1 and Rk1Rk2 compared to the control and Rk2. However, no apparent differences in reproduction occurred on Rk1 and/or Rk2 entries at 35ºC compared to the control, indicating parasitism increased on resistant entries at higher temperatures. / Master of Science in Life Sciences

La moléculture végétale est une alternative abordable et sécuritaire pour la production de protéines recombinantes d’intérêt clinique. Cependant, ces protéines sont souvent sujettes à la dégradation par les protéases endogènes dans les cellules végétales. Plusieurs stratégies ont été mises en place pour contrer ce phénomène, incluant notamment la co-expression d’inhibiteurs de protéases compagnons. Peu de données ont décrit toutefois comment la physiologie de la plante hôte, associée en particulier à son développement, a un impact sur l’expression et la protection d’une protéine d’intérêt. Dans ce projet, nous avons évalué les activités de protéases de types cystéine et aspartate dans des feuilles agroinfiltrées de la plante hôte Nicotiana benthamiana, en fonction de leur stade de développement. Nous démontrons que l’expression de l’inhibiteur SlCYS8 de tomate, actif contre les protéases de type cystéine, permet d’augmenter d’environ 40% le rendement en anticorps monoclonal C5-1, notre protéine modèle, dans les feuilles jeunes et matures où les activités protéolytiques sont moindres. Ces résultats prometteurs sur le plan appliqué font ressortir, en revanche, la complexité des processus biochimiques en cause et la pertinence de développer des stratégies complémentaires pour augmenter les teneurs en protéine recombinante. Dans cette optique, une nouvelle approche basée sur une diversion dirigée du métabolisme de défense de la plante hôte a été explorée. Nous démontrons qu’une activation du sentier de l’acide jasmonique induit dans les feuilles la production de protéines de stress, incluant des inhibiteurs de protéases de classes fonctionnelles variées; et une diminution de la teneur en RuBisCO, un contaminant majeur pendant la purification des protéines recombinantes. Sur cette base, nous avons adjoint à notre procédure de transfection un prétraitement des plantes au jasmonate de méthyle, une forme volatile de l’acide jasmonique. Ce prétraitement a permis d’une part de réduire considérablement les teneurs en RuBisCO dans les feuilles, et de doubler d’autre part le taux d’expression et le rendement en anticorps C5-1. Nos données appuient l’utilité éventuelle d’une manipulation du métabolisme de défense sur les rendements en protéine recombinante, complémentaire aux approches basées sur la co-expression d’inhibiteurs de protéases recombinants. / Plant molecular farming is an affordable and safe alternative to conventional expression animal and microbial systems for the production of clinically-useful recombinant proteins. However, these proteins are often susceptible to degradation by endogenous proteases of the host plant cells. Several strategies have been described to counter degradation processes including the co-expression of companion protease inhibitors. Few data, however, were available describing how the host plant’s physiology, especially its developmental stage, could impact the expression and protection of a clinically useful heterologous protein. In this study, we assessed changes in the activity of cysteine and aspartate proteases in the widely adopted expression host Nicotiana benthamiana, as a function of leaf age. We demonstrate that the expression of a tomato cysteine protease inhibitor, SlCYS8, allows to increase by nearly 40% the total yield of a model protein, the blood-typing C5-1 monoclonal antibody, in young and mature leaves where proteolytic activities are lower. Our results, while promising in practice, highlighted on the other hand the complexity of biochemical processes involved and the relevance of developing additional strategies to boost recombinant protein accumulation in planta. Towards this end, a new approach based on a deliberate diversion of the host plant defense metabolism was explored. We show that an activation of the jasmonic acid pathway in leaves induces the production of stress-related proteins, including protease inhibitors of various functional classes; and a significant decrease in the content of RuBisCO, a major contaminant during the purification of recombinant proteins. Based on this we included in our transfection procedure a pretreatment of plants with methyl jasmonate, a volatile form of jasmonic acid. This pretreatment significantly reduced RuBisCO levels in leaves, concomitant with a more than twofold increase of C5-1 yield in mature leaves. Our data highlight, overall, the potential of manipulating the host plant’s defense metabolism to increase heterologous protein yields, in complement to current protein protection strategies involving the co-expression of recombinant protease inhibitors.

The ecology and evolution of a species is often considered only within the context of pairwise interactions even though a species' distribution and abundance may be determined by interactions with many species within and between trophic levels. Multiple herbivores often share the same host and may interact indirectly by altering the relationships between herbivores, their host plants and their parasitoids. However, the relationships between parasitoids and herbivore hosts have typically been studied in isolation of other herbivore species. I examined how the outcomes of species interactions change when multiple relationships are considered. Chapter 1 examined the potentially conflicting selection pressures Manduca sexta exerts on Nicotiana tabacum (tobacco), since M. sexta has pollinating adults but herbivorous larvae. I demonstrated that high nectar amino acids do not affect floral visitation, but increased oviposition of herbivores on leaves. Thus, the relative costs and benefits of nectar rewards may depend on the community of pollinators and their life histories. In the remaining chapters I examined how feeding on tobacco by the aphid Myzus persicae altered the interactions between a parasitoid (Cotesia congregata) and its hornworm host (M. sexta). Chapter 2 demonstrated that aphids reduced hornworm abundance and parasitism. Changes in hornworm abundance were not due to density-dependent changes in moth oviposition, but the proportion of caterpillars attacked by parasitoids was inversely density dependent with hornworm density. Chapter 3 examined whether changes in hornworm abundance and parasitism reflected aphid-induced changes in host plant quality or volatile emissions. Aphids increased hornworm mortality, did not affect parasitoid performance, and increased parasitoid search time. In combination with Chapter 2, results suggest that aphids can mediate parasitoid-caterpillar interactions through changes in host plants that reduce hornworm survival and alter parasitoid behavior. Chapter 4 addressed how the outcome of interactions that are altered by aphids changed with spatial scale, and found no effect spatial scale on hornworm abundance and parasitism. In this system, aphids alter parasitoid-caterpillar interactions through changes in plant quality that reduce hornworm performance and abundance, and in turn, influence parasitoid attack. This work demonstrates that the outcome of multispecies interactions may not be predictable from pairwise interactions. / Ph. D.

Pepper and tomato, as two common, popular, and important vegetables grown worldwide, provide human beings with high quality fruit of flavor and aroma, and a high concentration of vitamins and antioxidants. Pepper and tomato production is frequently affected by various pathogens, including nematodes, fungi, and bacteria. Among those phytopathogens, Xanthomonas euvesicatoria (Xe) causes a severe bacterial spot (BS) disease on pepper and tomato. The BS disease could cause a loss of approximately 10% of the total crop yield in the world. Breeding tomato and pepper cultivars with improved BS disease resistance is one of the most important breeding goals. A better understanding of the virulence mechanism of Xe could help breeders design new strategies for resistance breeding. In this dissertation, we characterized the virulence and avirulence functions of four Xe Type Three Secretion Effectors (T3Es): Xe-XopQ, Xe-XopX, Xe-XopN, and Xe-avrRxo1. Xe-XopQ is a Xe T3E that functions as a determinant of host specificity. Here, we further explored the virulent and avirulent functions of Xe-XopQ. We identified another T3E Xe-XopX that could interact with XopQ and subsequently elicit the hypersensitive response in N. benthamiana in the Agrobacterium-mediated transient assay and Xe-mediated disease assay. The interaction is confirmed by bimolecular fluorescence complementation, co-immunoprecipitation and split luciferase assay. Intriguingly, we also revealed that XopX also interacts with multiple Xe T3Es including AvrBS2, XopN, XopB, and XopD in the co-IP assay. The virulent and avirulent functions of XopQ and AvrBS2 are compromised in the absence of Xe-XopX. Since XopX is conserved in diverse Xanthomonas spp., we speculate that Xe-XopX may have a general role required for the pathogenesis of Xe. Xe-XopN has been reported to be a T3E with virulence function via targeting host defense-related proteins, including atypical receptor-like kinase named TARK1 and a 14-3-3 protein to suppress the PAMPs (pathogen-associated molecular patterns) triggered immunity upon Xe colonization of tomato. In this study, we revealed additional virulence mechanisms of Xe-XopN, where Xe-XopN, is required for triggering the water-soaking symptom on Nicotiana benthamiana and pepper plants infected with Xe. In addition, we identified that XopN interacts with a transcription factor, NbVOZ, and represses the expression of NPR1, a key component of the basal defense. Therefore, XopN has a role in maintaining a water-affluent environment for better replication of Xe, and it can also interact with NbVOZ1/2 to regulate plant immunity. AvrRxo1, a T3E of Xanthomonas oryzae pv. oryzicola (Xoc), was previously identified to function as a NAD kinase. Here, we characterized a Xe T3E, Xe avrRxo1, that is a functional homologue of AvrRxo1, which is required for the full virulence of Xe to colonize the pepper and N. benthamiana plants. Overexpression of AvrRxo1 in bacterial or plant cells is toxic. Our group previously demonstrated AvrRxo1-ORF2 functions as an antitoxin that binds to AvrRxo1 to suppress its toxicity. In this study, we identified Xe4429 as the homologue of AvrRxo1-ORF2, which could interact with Xe-avrRxo1 to suppress its toxicity. We also revealed that Xe4429 could bind to the promoter of Xe-avrRxo1 and suppress its transcription. Therefore, we found Xe4429 encodes protein functions as an antitoxin and a transcription repressor in Xe bacterial cells. / Doctor of Philosophy / Peppers and tomatoes are two of the most important vegetables grown worldwide, providing humans with high quality of flavor and aroma, vitamins, and antioxidants. The pepper and tomato production is frequently threatened by various pathogens, including nematodes, fungi, and bacteria. Among those phytopathogens, Xanthomonas euvesicatoria (Xe) causes a severe bacterial spot (BS) disease on peppers and tomatoes. The BS disease can be easily identified due to the appearance of the dark, irregular, water-soaked areas on the leaf, which can cause approximately 10% loss of the total yield of peppers and tomatoes. Breeding tomato and pepper cultivars with improved BS disease resistance is one of the most critical breeding goals. A better understanding of the virulence mechanism of Xe could help breeders to design new strategies for resistance breeding. In my seminar, I will discuss the virulence and avirulence functions of Xe type three secretion (T3S) effectors: Xe XopN, Xe XopQ, and Xe XopX. In my study, I identified Xe XopN is a key factor that regulates the development of the water-soaking symptom on pepper plants infected with Xe. In addition, we revealed Xe XopN interacts with a transcription factor NbVOZ to regulate the expression of NbNPR1 and PR1 genes expression, which may also contribute to the development of water-soaking phenotype. In addition, I identified that Xe XopN could interact with a transcription factor, NbVOZ, and represses the expression of NbNPR1, a key component of the basal defense, and the pathogenesis-related gene PR1. Therefore, Xe XopN has a role in regulating a water-affluent environment to promote bacterial proliferation in the infected plant tissue. Xe XopQ is a Xe T3S effector that functions as a determinant of host specificity. In my study, I identified another T3S effector Xe XopX that could interact with Xe XopQ to trigger the defense response in Nicotiana benthamiana. I also confirmed Xe XopQ physically interacts with Xe XopX inside of plant cells by using bimolecular fluorescence complementation, co-immunoprecipitation and split luciferase assay. Intriguingly, Xe XopX could also interact with multiple Xe T3Es including AvrBS2 in a co-IP assay. The virulence and avirulent functions of Xe XopQ and AvrBS2 are compromised in the absence of Xe XopX.

Pheromone lures and trap crops are appealing pest management tools that use insect and/or plant volatiles to reduce pest populations on crops of interest. Generating pheromone-emitting trap plants may allow for a continuing and highly-specific attraction of insect pests without repeated and costly application of synthetic pheromones. These trap plants may also be used to develop area-wide pest management strategies. As a proof-of-principle study we tested the possibility of producing the pheromone of the crucifer flea beetle Phyllotreta cruciferae in transgenic plants. P. cruciferae is an important pest of Brassica crops. In the presence of a host plant, males emit an aggregation pheromone, which attracts both males and females. Himachaladiene, a sesquiterpene, has been identified as a key component of the aggregation pheromone of P. cruciferae. In a close relative, Phyllotreta striolata, the compound is synthesized by a two-step pathway with an isoprenyl diphosphate synthase (PsIDS3) making (Z,E)-farnesyl diphosphate (FPP), which is converted by a terpene synthase (PsTPS1) to himachaladiene. Transient transformation of N. benthamiana with PsIDS3-TPS1 co-localized to the plastid resulted in the emission of himachaladiene and other known PsTPS1 products. Daily emissions of himachaladiene were approximately 1 µg per plant, which is six-fold higher than emissions from individual male flea beetles. Stable transformation of Arabidopsis thaliana with the same vector construct resulted in transgenic plants that expressed PsTPS1 and PsIDS3 transcripts, but no himachaladiene or other PsTPS1 products were present in volatile collections or leaf extracts of these plants. Moreover, no PsTPS1 enzyme activity was observed, indicating that post-transcriptional/translational effects prevent proper expression or targeting of functional PsIDS3 and/or PsTPS1 proteins in A. thaliana. Overall, this study demonstrates that the key component of the P. cruciferae aggregation pheromone, himachaladiene, can be transiently produced and emitted in a plant system at rates that are biologically relevant for insect attraction. However, further work is required for the stable production of the pheromone in plants. In addition, preliminary results are presented for the development of simple two-choice arenas that may allow for assessment of the movement of beetles toward host plant leaf tissue. This work can inform future efforts in developing methods for the economic production of himachaladiene in a plant system or the establishment of transgenic plants for the production and deployment of himachaladiene in a field setting. / Master of Science / The crucifer flea beetle is an important pest of vegetable and oilseed Brassica crops such as broccoli, cabbage and canola. Feeding by beetles has its greatest impact on crop health and yield in the early spring, when adult beetles emerge from overwintering sites and feed on newly- emerging Brassica seedlings. Currently these insects are controlled using broad spectrum insecticides. A general awareness of the negative aspects of insecticides drives the search for alternative pest management strategies that could diversify our management strategies and reduce reliance on insecticides. Previous work has found that the crucifer flea beetle navigates to its host plants, in part, through plant-emitted volatiles. After locating the plant host, males emit a volatile aggregation pheromone that when blended with host plant volatiles increases attraction. Here work towards the development of a specialized trap crop is presented. Plants were engineered to emit a key component of the crucifer flea beetle aggregation pheromone. In an engineered non-host plant, Nicotiana benthamiana, transient production of the aggregation pheromone was established. However, in an engineered Brassica plant, Arabidopsis thaliana, no aggregation pheromone was detected despite evidence of the presence and expression of the required biosynthetic genes for its production. A discussion on alternative engineering strategies for A. thaliana is presented. In addition, preliminary results are presented for the development of a simple behavior assay to assess the attraction of beetles toward different smells. This work can inform future efforts aimed at developing methods for the economic production of the aggregation pheromone in a plant system or the establishment of plants for the production and deployment of the aggregation pheromone in a field setting.

N-methylputrescine oxidase (MPO) is a member of the diamine oxidase (DAO) class of enzymes believed to be responsible for synthesis of the alkaloid nicotine in the roots of Nicotiana tabacum (Mizusaki et al., 1972). A purportedly pure MPO protein from tobacco root culture extracts was used to generate immune antiserum in rabbits (McLauchlan et al., 1993). In an attempt to clone a cDNA encoding MPO, we used this antiserum to screen a tobacco cDNA expression library. Unexpectedly, two previously unreported genes with strong homology to members of a gene family encoding S-adenosylhomocysteine hydrolase (SAHH) in N. sylvestris and a gene encoding SAHH in N. tabacum were cloned instead. SAHH is an enzyme of the S-adenosylmethionine (SAM) recycling pathway, which also includes SAM synthetase (SAMS) and methionine synthase (MS). These results led to the hypothesis of a multi-enzyme complex, or metabolon, of at least one member of the nicotine biosynthesis pathway, i.e., MPO, and at least one member of the SAM recycling pathway, i.e., SAHH, during nicotine biosynthesis. Metabolons are stable noncovalent complexes in cells that ensure sufficient passage of the product of one enzyme reaction to the next enzyme in the pathway via a "channel" without equilibrating with the bulk solution (Ovádi, 1991). My research employed co-immunoprecipitation studies to determine if other SAM recycling enzymes are associated in a complex with MPO and SAHH, as well as Northern and Western blot analyses to determine if the genes encoding SAM recycling pathway enzymes are coordinately regulated during nicotine biosynthesis. Our results indicate that nicotine biosynthesis-inducing conditions result in differential mRNA accumulation patterns of the three enzymes of the SAM recycling pathway, although to different extents. However, protein levels of SAM recycling pathway members do not appear to reflect the differential mRNA accumulation patterns. We have firmly established an association of SAHH and an enzyme with DAO activity, purportedly MPO. If the enzyme is proven to be MPO, then our data would constitute the first documentation of an alkaloid metabolon. Finally, using a degenerate primer PCR approach, we have cloned a 986-bp gene fragment with homology to copper amine oxidases, the class to which MPO belongs. / Master of Science

Les systèmes d’expression végétaux sont utilisés couramment pour la production hétérologue de protéines recombinantes complexes. Des contraintes biochimiques dans le système de sécrétion cellulaire, comme l’abondance de protéases peu spécifiques ou des variations de pH d’un organite à l’autre, compromettent toutefois l’expression de plusieurs protéines d’intérêt. Des approches ont été développées pour améliorer l’environnement intracellulaire dans la plante hôte de manière à accroître la qualité et le rendement des protéines sécrétées. Dans ce projet, nous avons évalué l’impact de l’homéostasie du pH dans le système de sécrétion cellulaire des feuilles du Nicotiana benthamiana sur l’expression et la stabilité des protéines recombinantes. Nous démontrons le potentiel du canal ionique M2 du virus de l’Influenza comme nouvel outil pour augmenter le pH des compartiments acides du système de sécrétion cellulaire, une approche éventuellement utile pour la stabilisation des protéines sensibles aux milieux acides. En lien avec l’influence bien documentée du pH sur l’activité des protéases cellulaires, nous montrons ensuite qu’une modification du pH induite par l’expression du canal M2 influence les profils de dégradation de protéines de fusion sensibles à la protéolyse, des observations qui confirment l’impact du pH sur l’activité protéolytique des cellules végétales et qui suggèrent le potentiel du canal ionique M2 comme protéine accessoire pour augmenter la stabilité et le rendement des protéines recombinantes in planta. Finalement, nous abordons l’impact du canal ionique M2 sur l’expression des protéines endogènes à l’échelle de la cellule. Nous démontrons qu’une altération du pH dans le système de sécrétion en réponse à l’expression du canal ionique a des effets étendus sur le protéome foliaire, affectant la teneur de protéines retrouvées dans plusieurs compartiments cellulaires incluant les chloroplastes, le cytosol et la vacuole. Nous rapportons aussi l’établissement d’un ‘protéome hybride’ dans les plantes exprimant M2, composé de protéines caractéristiques aussi bien de plantes témoins non infectées que de plantes agroinfectées exprimant activement des protéines de défense. En résumé, nos données mettent en évidence le rôle de l’homéostasie du pH sur le protéome des cellules végétales et l’impact significatif du gradient de pH dans le système de sécrétion cellulaire sur la stabilité et le rendement des protéines recombinantes sensibles à l’acidité ou à la protéolyse. / Plant expression systems are commonly used for the heterologous production of complex recombinant proteins. However, biochemical conditions in the plant cell secretory pathway, such as the presence of poorly-specific proteases or pH variations from one organelle to another, impair the expression of several potentially useful proteins. Approaches have been developed to improve the cellular environment of the host plant in such a way as to increase the quality and yield of secreted recombinant proteins. In this project, we assessed the impact of pH homoeostasis in the leaf cell secretory pathway of wild tobacco Nicotiana benthamiana on the expression and stability of recombinant proteins. We demonstrate the potential of Influenza virus proton channel M2 as a new tool to increase pH in acidic compartments of the cell secretory pathway, eventually useful to stabilize acid-labile recombinant proteins. In line with the well-established influence of pH on cell protease activities, we then show that pH modification induced by the expression of the M2 channel influences the degradation profile of fusion proteins susceptible to proteolysis, thus confirming the impact of pH on protease activities in plant cells and highlighting the potential of M2 as an accessory protein to increase the stability and yield of recombinant proteins in planta. Finally, we describe the impact of the M2 proton channel on the expression of endogenous proteins at the cellular scale. We show that pH alteration in the secretion system upon M2 expression has cell-wide effects on the leaf proteome, affecting the content of proteins found in various cell compartments including the chloroplast, the cytosol and the vacuole. We report the establishment of a ‘hybrid proteome’ in leaf cells expressing the proton channel, composed of protein clusters characteristic of both control, non-infected plants and agroinfected plants actively expressing defense-related proteins. Overall, our data highlight the central role of pH homeostasis on the proteome of plant cells and the strong impact of pH gradient in the cell secretory pathway on the stability and yield of acidic pH-labile and protease-susceptible recombinant proteins.

La cryptogéine, sécrétée par l’oomycète Phytophthora cryptogea, est un éliciteur protéique des réactions de défense qui active chez le tabac un ensemble d’événements de signalisation conduisant à la mise en place d’une mort cellulaire de type réponse hypersensible et d’une résistance systémique acquise. La caractérisation de la modulation de l’activité de kinases cytosoliques, dont SIPK et WIPK, par des événements de phosphorylation en réponse à la cryptogéine, traduit la place majeure que tiennent les modifications post-traductionnelles dans la cascade de signalisation induite dans les cellules de tabac en réponse à la cryptogéine. Il s’avère que la signalisation cellulaire induite par la cryptogéine, et impliquant ces protéines kinases, converge entre autre vers le noyau à travers la modulation de l’activité d’éléments nucléaires par phosphorylation. Dans ce contexte, d’importants travaux de purification/séquencage, visant à identifier les protéines nucléaires cibles de ces activités kinases, ont permis d’identifier deux isoformes redondantes d’histones désacétylases de type 2 nommés NtHD2a et NtHD2b qui sont rapidement phosphorylées en réponse à la cryptogéine dans les cellules de tabac.Ce travail de thèse s’inscrit dans l’étude du rôle de NtHD2a/b dans l’établissement du processus de mort cellulaire des cellules de tabac et de la RH in planta en réponse à la cryptogéine. Par des approches de pharmacologie ainsi que des approches de surexpression ou d’invalidation de l’expression de NtHD2a/b chez le tabac, nous avons d’une part confirmé l’implication de NtHD2a/b en tant que régulateurs négatifs de la mort cellulaire induite par la cryptogéine ou d’autres élicitines, et d’autre part mieux appréhendé les événements de la cascade de signalisation prépondérants dans l’établissement de cette mort cellulaire. Les mécanismes moléculaires sous-jacents à la mise en place de la mort cellulaire apparaissent complexes et semblent notamment impliquer la modulation de l’expression de gènes de défense, la synthèse de novo de protéines ainsi que l’activation de protéines kinases, dont notamment WIPK et SIPK.Des travaux relatifs à l’étude des événements de (dé)/acétylation dans les cellules de tabac traitées par la cryptogéine et invalidées dans l’expression de NtHD2a/b suggèrent le concours de modifications post-traductionnelles de protéines nucléaires telles que l’acétylation dans la mise en place de la mort cellulaire induite par la cryptogéine chez le tabac. / Cryptogein, which is secreted by the oomycete Phytophthora cryptogea, is a proteinaceous elicitor of plant defense reactions that activates a set of signaling events leading to the hypersensitive response and to systemic acquired resistance. Although the early cytosolic signaling events induced by cryptogein are well described, the only nuclear events characterized to date are the variations in free calcium concentrations and defense-related gene expression. The characterization of the activation of cytosolic protein kinases, including WIPK and SIPK, by phosphorylation in response to cryptogein highlights the key-role played by posttranslational modifications in cryptogein-induced signaling events in tobacco cells. In this context, purification/sequencing approaches revealed that two redundant isoforms of type-2 nuclear histone deacetylases, NtHD2a and NtHD2b, were rapidly phosphorylated in cryptogein-treated tobacco cells.This thesis work is part of a comprehensive study of the role of NtHD2a/b in the establishment of the cell death process in tobacco cells and of the hypersensitive response in planta, in response to cryptogein. By using a pharmacological approach and overexpression and RNA interference-based approaches, we confirmed the involvement of NtHD2a/b as negative regulators of elicitin-induced cell death and we achieved a better understanding of cell death signaling events. The molecular events that underly the cell death process appear particularly complex and seem to involve the modulation of defense-related gene expression, de novo protein synthesis and protein kinase activation such as WIPK and SIPK.The study of (de)/acetylation events in tobacco cells treated by cryptogein and invalidated in NtHD2a/b expression suggests a role for posttranslational modifications in cryptogein-induced cell death.

A biologia da reprodução de plantas é um campo de grande interesse, já que a maioria dos alimentos consumidos pelo homem é composta de partes reprodutivas das plantas (frutos e sementes). O pistilo é o órgão reprodutivo feminino, composto de estigma, estilete e ovário. Devido à importância central do pistilo no sucesso da reprodução de plantas, faz-se necessário um melhor conhecimento dos genes e processos que regulam seu desenvolvimento e funcionamento. Estudos comparativos da expressão gênica nos órgãos vegetativos e reprodutivos de Nicotiana tabacum revelaram genes de expressão preferencial nos órgãos reprodutivos, entre eles alguns codificando proteínas de função ainda desconhecida. Um destes genes foi caracterizado e denominado SCI1 (Stigma/style Cell-cycle Inhibitor 1), por apresentar um papel importante no desenvolvimento do estigma/estilete, atuando como um inibidor de ciclo celular tecido-específico (DePaoli et al., 2011). O presente trabalho teve como objetivo estudar os mecanismos moleculares pelos quais NtSCI1 regula o ciclo celular, investigando seus parceiros de interação. Em um ensaio de pull-down, utilizando-se extrato proteico nuclear de estigmas/estiletes de N. tabacum, vários putativos reguladores de ciclo celular foram identificados, sendo a interação entre NtSCI1 e NtCDKG;2 confirmada por BiFC e localizada no nucléolo. Uma biblioteca de cDNAs de estigmas/estiletes de N. tabacum, no sistema de duplo-híbrido de levedura, foi construída com sucesso. O screening desta biblioteca, utilizando BD-NtSCI1 como \"isca\", permitiu a identificação de vários parceiros de interação com NtSCI1, entre eles: uma helicase de RNA DEAD-BOX, a proteína 14-3-3D2, dois fatores de transcrição (HOMEOBOX-22 e STOREKEEPER), um fator de splicing portador do domínio SWAP, uma quinase de adenosina e uma transposase. As interações entre NtSCI1 e os três primeiros parceiros citados já foram confirmadas por BiFC (observadas no núcleo e nucléolo) e a interação entre NtSCI1 e Nt14-3-3D2 foi confirmada também por co-imunoprecipitação. O envolvimento de NtSCI1 com a regulação do ciclo celular foi corroborado pela interação entre NtSCI1 e a proteína NtCICLINA-L1 (subunidade regulatória de CDKG;2), confirmada por duplo-híbrido e por BiFC, no nucléolo. A interação entre NtSCI1 e NtCICLINA-RELATED também foi confirmada por BiFC. Para entender a dinâmica de NtSCI1 no nucléolo, foi estudada a localização subcelular da proteína de fusão NtSCI1-GFP durante as fases do ciclo celular. NtSCI1-GFP foi observada no nucléolo de células BY-2 em interfase e prófase, desaparecendo na metáfase e anáfase e reaparecendo no nucléolo no final da telófase, mostrando que a presença de NtSCI1 na célula é controlada pelo ciclo celular. A construção de uma primeira versão do interactoma de NtSCI1 mostrou seu envolvimento direto e indireto com proteínas relacionadas ao metabolismo de RNAs, controle da transcrição e regulação do ciclo celular. Estes resultados sugerem que NtSCI1 possa atuar no controle do ciclo celular de forma não canônica, por meio de múltiplos processos paralelos que interconectam aspectos da regulação da transcrição e o processamento de RNAs com o controle do ciclo celular. / The biology of plant reproduction is a field of great interest, since most of the food consumed by humans is composed of reproductive parts of plants (fruits and seeds). The pistil is the female reproductive organ, composed of stigma, style and ovary. Due to the central importance of the pistil in the success of plant reproduction, a better knowledge of the genes and processes that regulate pistil development and function is necessary. Comparative studies of gene expression in vegetative and reproductive organs of Nicotiana tabacum have revealed genes preferentially expressed in the reproductive organs, among them some encoding proteins of unknown function. One of these genes was characterized and denominated SCI1 (Stigma/style Cell-cycle Inhibitor 1), since it has an important role in stigma/style development, acting as a tissue-specific cell-cycle inhibitor (DePaoli et al., 2011). The objective of the present work was to study the molecular mechanisms through which NtSCI1 regulates the cell cycle investigating its interaction partners. In a pull-down assay, using nuclear protein extracts from N. tabacum stigmas/styles, several putative cell cycle regulators were identified. Among them, the interaction between NtSCI1 and NtCDKG;2 was confirmed by BiFC and localized in the nucleolus. A N. tabacum stigma/style cDNA library in the yeast two-hybrid system was successfully constructed. The screening of this library, using BD-NtSCI1 as bait, allowed the identification of several NtSCI1 interaction partners, among them: a DEAD-BOX RNA helicase; the 14-3-3D2 protein; two transcription factors (HOMEOBOX-22 and STOREKEEPER); a splicing factor containing a SWAP domain; an adenosine kinase; and a transposase. The interactions between NtSCI1 and the first three mentioned partners have already been confirmed by BiFC (observed in the nucleus and nucleolus) and the interaction between NtSCI1 and Nt14-3-3D2 was also wconfirmed by co-immunoprecipitation. The NtSCI1 involvement in cell cycle regulation was corroborated by the interaction between NtSCI1 and the NtCYCLIN-L1 (a regulatory subunit of CDKG;2), which was confirmed by two-hybrid and BiFC in the nucleolus. The interaction between NtSCI1 and NtCYCLIN-RELATED was also confirmed by BiFC. To understand the dynamics of NtSCI1 in the nucleolus, the subcellular localization of the fusion protein NtSCI1-GFP was studied during the different cell cycle phases. NtSCI1-GFP was observed in the nucleolus of BY-2 cells at interphase and prophase, disappearing at metaphase and anaphase and reappearing in the nucleolus at the end of telophase, showing that NtSCI1 presence in the cell is controlled by the cell cycle. The construction of the first version of NtSCI1 interactome showed its direct and indirect involvement with proteins related to RNA metabolism, transcription control and cell cycle regulation. These results suggest that NtSCI1 may act in cell cycle control in a non-canonical way, through multiple parallel processes interconnecting aspects of transcription regulation, RNA processing and cell cycle control.

A eficiência da reprodução sexual de plantas depende do correto desenvolvimento dos órgãos sexuais: estame e pistilo. Mecanismos moleculares complexos controlam a proliferação e expansão celular que resultam no correto desenvolvimento destes órgãos. Em nosso laboratório foi identificado um gene preferencialmente expresso no pistilo de Nicotiana tabacum, o gene 092H06. Este gene codifica uma pequena proteína de 68 aminoácidos, e função desconhecida. Análises anteriores sugerem que o produto proteico do gene 092H06 seja responsável por inibir o processo de expansão celular nos órgãos reprodutivos (Brito,2010). Para compreender o papel deste gene, no desenvolvimento do pistilo, foram realizados experimentos de qRT-PCR para determinar se os níveis de expressão de genes para -expansina, -expansina, ciclina B1.2 e actina, ligados aos processos de divisão e expansão celular, em plantas transgênicas de silenciamento e superexpressão do gene 092H06. Foram realizadas análises morfológicas nos estigmas/estiletes e ovários das plantas transgênicas de segunda geração (T2), por microscopia óptica. Os resultados mostram uma tendência de aumento no volume das células tanto nas plantas transgênicas de silenciamento, como nas de superexpressão. Entretanto, nas plantas de silenciamento ocorreu um aumento visível das estruturas reprodutivas, o que não foi observado nas plantas de superexpressão. Adicionalmente, foram realizados experimentos de citometria de fluxo, para verificar a ocorrência de endorreduplicação. Os resultados mostraram que não ocorreu endorreduplicação nas células das plantas transgênicas. No screening de uma biblioteca de duplo híbrido, usando 092H06 como isca, foram encontrados 4 candidatos a parceiros de interação: 1) biotin/lipolyl attachmente domain-containing protein; 2) unknown protein; 3) trypsin proteinase inhibitor precursor e 4) RING/U-box. Para auxiliar no estudo da função do gene 092H06, a proteína recombinante 092H06-Histag foi produzida com sucesso, na forma solúvel, em E. coli. Os resultados alcançados neste trabalho contribuem para avançar o conhecimento sobre este novo gene expresso nos órgãos reprodutivos das plantas. / The efficiency of plant sexual reproduction depends on the correct development of the sexual organs: stamen and pistil. Complex molecular mechanisms control cell proliferation and expansion that result in the correct development of these organs. In our laboratory a gene preferentially expressed in Nicotiana tabacum pistil has identified, the 092H06 gene. This gene encodes a small protein of 68 amino acids of unknown function. Previous analyzes suggest that the protein product of the gene 092H06 is responsible for inhibiting the cell expansion process in the reproductive organs (Brito, 2010). To understand the role of this gene in pistil development, experiments of qRT-PCR to determinate the expression levels of the -expansins, -expansins, cyclin B1.2 and actin, genes which connected to the cell division and expansion processes, were carried out on transgenic plants silencing and overexpressing the 092H06 gene. Morphological analyzes on stigmas/styles and ovaries of second generation (T2) transgenic plants were performed by optical microscopy. The results show a tendency to increased cellular volume on the silencing transgenic plants, as well as on the overexpressing plants. However, in the silencing plants there was a visible increase of the reproductive structures, what has not been observed on the overexpressing plants. Additionally, flow cytometry experiments were carried out to verify the occurrence of endoreduplication. The results showed that no endoreduplication has occurred on the cells of the transgenic plants. The screening of a yeast two-hybrid assays, using 092H06 as bait, has found 4 interaction partners candidates: 1) biotin/lipolyl attachment domain-containing protein; 2) unknown protein; 3) trypsin proteinase inhibitor precursor and 4) RING/U-box. To assist the study of the 092H06 function, the recombinant 092H06-HIStag protein has been produced with success, in the soluble form, in E.col. The results obtained in this work contribute to advance the knowledge of this novel gene expressed on the plant reproductive organs.