Soybean is a dominant oilseed cultivated worldwide for its use in multiple sectors such as food and feed industries, animal husbandry, cosmetics and pharmaceutical sectors, and more recently, in production of biodiesel. Increasing demand of soybean, changing environmental conditions, and evolution of pathogens pose challenges to soybean production in limited acreage. Genetic research is the key to ensure the continued growth in soybean production, with enhanced yield and quality, while reducing the losses due to diseases and pests. This research is focused on the understanding of transcriptional regulation of two economically important agronomic traits of soybean: low seed phytic acid and resistance to Soybean mosaic virus (SMV), using the 'transcriptomics' and 'genomics' approaches. The low phytic acid (lpa) soybean is more desirable than conventional soybean, as phytic acid is an anti-nutritional component of seed and is associated with phosphorus pollution. Despite the eco-friendly nature of the lpa soybean, it shows poor emergence, which reduces soybean yield. This research is mainly focused on addressing the impact of lpa-causing mutations on seed development, which is suspected to cause low emergence in lpa soybeans. The differences in transcriptome profiles of developing seeds in lpa and normal phytic acid soybean are revealed and the biological pathways that may potentially be involved in regulation of seed development are suggested. The second research project is focused on Rsv3-type resistance, which is effective against most virulent strains of Soybean mosaic virus. The Rsv3 locus, which maps on to soybean chromosome 14, contains 10 genes including a cluster of coiled coil-nucleotide binding-leucine rich repeat (CC-NB-LRR) protein-encoding genes. This dissertation employed a comparative sequencing approach to narrow down the list of Rsv3 gene candidates to the most promising CC-NB-LRR gene. The evidence provided in this study clearly indicates a single CC-NB-LRR gene as the most promising candidate to deliver Rsv3-type resistance. / Ph. D.

Soybean mosaic virus (SMV) is the most common member of the viral genus Potyvirus to infect soybeans (Glycine max [L.] Merr.) worldwide. SMV has been traditionally controlled by the deployment of single dominant, strain specific resistance genes, referred to as Rsv genes. Rsv1 is the most widely used form of SMV resistance with nine different alleles conferring resistance only to the lower numbered less virulent strains, G1 to G3. Rsv3 gives resistance to higher numbered more virulent strains G5 to G7. Soybean lines containing Rsv4, are resistant to all seven currently recognized North American SMV strains. In this study, the recently released soybean whole genome sequence was used to design molecular markers for fine mapping Rsv3 to a ~150 kb genomic region containing four coiled-coil nucleotide-binding leucine-rich repeat proteins. In a related study a large population segregating at the Rsv3 locus was screened for resistance to facilitate future characterization of this region. The markers identified in this study will allow for more accurate marker-assisted selection of Rsv3. / Master of Science

Internalin J (InlJ) gehört zu der Klasse der bakteriellen, cysteinhaltigen (leucine-rich repeat) LRR Proteine. Bei den Internalinen handelt es sich um meist invasions-assoziierte Proteine der Listerien. Die LRR-Domäne von InlJ ist aus 15 regelmäßig wiederkehrenden, stark konservierten Sequenzeinheiten (repeats, 21 Aminosäuren) aufgebaut. Ein interessantes Detail dieses Internalins ist das stark konservierte Cystein innerhalb der repeats. Daraus ergibt sich eine ungewöhnliche Anordnung von 12 Cysteinen in einem Stapel. Die Häufigkeit von Cysteinen in InlJ ist für ein extrazelluläres Protein von L. monocytogenes außergewöhnlich, und die Frage nach ihrer Funktion daher umso brennender. Im Vergleich zum ubiquitären Vorkommen der sogenannten repeat-Proteine in der Natur sind Studien zu ihrer Stabilität und Faltung nicht äquivalent vertreten. Die zentrale Eigenschaft der repeat-Proteine ist ihr modularer Aufbau, der durch einfache Topologie gekennzeichnet ist und auf kurzreichenden Wechselwirkungen basiert. Diese Topologie macht repeat-Proteine zu idealen Modellproteinen, um die stabilitätsrelevanten Wechselwirkungen zu separieren und zuzuordnen. In der vorliegenden Arbeit wurde die Faltung und Entfaltung von InlJ umfassend charakterisiert und die Relevanz der Cysteine näher beleuchtet. Die spektroskopische Charakterisierung von InlJ zeigte, dass dessen Faltungszustand durch zwei Tryptophane im N- und C-Terminus fluoreszenzspektroskopisch gut zugänglich ist. Die thermodynamische Stabilität wurde mittels fluoreszenz-detektierten, Guanidiniumchlorid-induzierten Gleichgewichtsexperimenten bestimmt. Um die kinetischen Eigenschaften von InlJ zu erfassen, wurden die Faltungs- sowie die Entfaltungsreaktion spektroskopisch untersucht. Die Identifizierung der produktiven Faltungsreaktion war lediglich durch die Anwendung des reversen Doppelsprungexperiments möglich. Die Auswertung erfolgte nach dem Zweizustandsmodell, wonach die Faltung dem „Alles-oder-Nichts“ Prinzip folgt. Die Gültigkeit dieser Annahme wurde durch die kinetische Charakterisierung bestätigt. Es wurde sowohl in den Gleichgewichtsexperimenten als auch in den kinetisch erhaltenen Daten eine hohe freie Stabilisierungsenthalpie festgestellt. Die hohe Stabilität von InlJ geht mit hoher Kooperativität einher. Die kinetischen Daten zeigen zudem, dass die hohe Kooperativität hauptsächlich der Faltungsreaktion entstammt. Der Tanford-Wert von 0.93 impliziert, dass die Oberflächenänderung während der Faltung bereits zum größten Teil erfolgt ist, bevor der Übergangszustand ausgebildet wurde. Direkte strukturelle Informationen über den Übergangszustand wurden mit Hilfe von Mutationsstudien erhalten. Zu diesem Zweck wurden 12 der 14 Cysteine gegen ein Alanin ausgetauscht. Die repeats 1 bis 11 von InlJ beinhalten jeweils ein Cystein, deren Anordnung eine Leiter ergibt. Deren Substitutionen haben einen vergleichbar destabilisierenden Effekt auf InlJ von durchschnittlich 4.8 kJ/mol. Die Verlangsamung der Faltung deutet daraufhin, dass die Interaktionen der repeats 5 bis 11 im Übergangszustand bereits voll ausgebildet sind. Demnach liegt bei InlJ ein zentraler Faltungsnukleus vor. Im Rahmen dieser Promotionsarbeit wurde eine hohe Stabilität und ein stark-kooperatives Verhalten für das extrazelluläre Protein InlJ beobachtet. Diese Erkenntnisse könnten wichtige Beiträge zur Entwicklung artifizieller repeat-Proteine leisten, deren Verwendung sich stetig ausweitet. / Internalin J (InlJ) is a member of the family of bacterial cysteine-containing leucine-rich repeat (LRR) proteins. Internalins are invasion-associated surface proteins of Listeria monocytogenes. The LRR domain of InlJ consists of 15 repeating units, which are arranged in tandem. The consensus sequence consists of 21 residues. Interestingly, a leucine residue which is highly conserved among the Internalins is replaced by cysteine. This results in a continuous cysteine ladder of 12 repeats. This frequency of cysteines is remarkable for an extracellular protein of L. monocytogenes. Stability and folding of repeat proteins are not equivalently studied considering their ubiquitous distribution in nature. Their modular structure results in simple topology and is dominated by short-range interactions. These characteristic features of repeat proteins facilitate the separation and identification of stabilizing interactions, making repeat proteins to ideal model systems for folding studies. In this work the folding and unfolding of InlJ has been extensively characterized, shedding light on the relevance of the cysteines. Two tryptophans located in the N- and C-terminus allowed monitoring the folding state of the entire protein via fluorescence. Thermodynamic stability was therefore derived by guanidinium chloride induced equilibrium experiments. Furthermore, the chemically induced unfolding and folding reactions were characterized with respect to their kinetics. Interrupted refolding experiments were essential for tracking the productive folding reaction of InlJ. Analysis of the kinetic and equilibrium data leads to the conclusion that the results are compatible with a two-state model. The study presented here reveals high stability of the protein InlJ in conjunction with high cooperativity. Kinetic data disclosed the origin of high cooperativity in the folding reaction; with a Tanford value of about 0.93. This high value implicates that the major change of the accessible surface area occurs before the transition state is formed. Mutational studies provided more detailed structural information about the transition state. 12 of 14 cysteine residues were mutated to alanine for this purpose. The cysteines in repeats 1 to 11 stack over each other and form a ladder of reduced cysteines. The substitution of one of these cysteines has an average destabilizing effect of 4.8 kJ/mol. The deceleration of the folding reaction by the substitution shows that repeats 5 to 11 are already fully structured in the transition state, pointing to a central nucleus in the folding of the LRR-protein InlJ. The extracellular protein InlJ reveals extreme stability and high cooperativity. The insights into the folding of this LRR motif could facilitate the design of further artificial repeat proteins.

Master of Science / Department of Plant Pathology / Karen A. Garrett / Ecological clines often result in gradients of disease pressure in natural plant communities, imposing a gradient of selection on disease resistance genes. We describe the diversity of a resistance gene homolog in natural populations of the dominant tallgrass prairie grass, Andropogon gerardii, across a precipitation gradient ranging from 47.63 cm/year in western Kansas to 104.7 cm/year in central Missouri. Since moisture facilitates infection by foliar bacterial pathogens, plants along this precipitation gradient will tend to experience heavier bacterial disease pressure to the east. In maize, the gene Rxo1 confers resistance to the pathogenic bacterium Burkholderia andropogonis. Rxo1 homologs have been identified in A. gerardii and B. andropogonis is known to infect natural populations of A. gerardii. The spatial genetic structure of A. gerardii was assessed from central Missouri to western Kansas by genotyping with AFLP markers. Samples were also genotyped for Rxo1 homologs by amplifying an 810 base pair region of the leucine-rich repeat and digesting with restriction enzymes. We compared Rxo1 homolog diversity to AFLP diversity across different spatial scales. Genetic dissimilarity based on AFLP markers was lower than would have occurred by chance at distances up to 30 m, and different prairies were more dissimilar than would have occurred by chance, but there was not a longitudinal trend in within-prairie dissimilarity as measured by AFLP markers. Dissimilarity of the Rxo1 homologs was higher in the east suggesting the presence of diversifying selection in the more disease-conducive eastern environments.

Au cours du développement des végétaux, de l’établissement de l’identité cellulaire des premiers organes au guidage du tube pollinique, la communication cellule à cellule est d’une importance capitale. En réponse, les voies de signalisation moléculaires sont élaborées pour la perception d’un signal extérieur et la transduction en une réponse génique via une cascade intracellulaire. Les récepteurs kinases font partie des protéines perceptrices des stimuli et constituent chez les plantes une catégorie de protéines avec une occurrence considérable, mais dont très peu d’informations détaillées sont disponibles à ce jour. Une famille de récepteurs kinases chez Arabidopsis thaliana, AtORK11 (Arabidopsis thaliana Ovule Receptor Kinase 11), a été identifiée par orthologie à un récepteur spécifique aux ovaires chez une solanacéee sauvage, Solanum chacoense. La fonction présumée de cette famille de récepteurs kinases de type leucine-rich repeat, suggérée par son patron d’expression, implique les événements relatifs au développement des gamétophytes et à la reproduction. Afin de caractériser la fonction des quatre gènes de la famille (AtORK11a, AtORK11b, AtORK11c et AtORK11d) une stratégie d’analyse de mutants d’insertion de l’ADN-T et d’évaluation du mode d’action par complémentation bimoléculaire par fluorescence (BiFC) a été entreprise. Aucune fonction précise n’a pu être attribuée aux doubles mutants d’insertion, par contre la surexpression d’une construction dominante négative indique un rôle dans le développement gamétophytique. Il a aussi été démontré que les quatre récepteurs peuvent interagir par homodimérisation aussi bien que par hétérodimérisation. Une hypothèse de redondance fonctionnelle est ainsi mise à jour parmi la famille des gènes AtORK11. / Cell to cell communication is paramount during plant developmental processes, from cellular identity in early organogenesis to pollen tube guidance. In response to this requirement, molecular cell signalling is used to perceive an external signal and transduce the response by an intracellular signalling cascade leading to specific gene activation. The sensing protein is typically a receptor kinase, which will transduce the stimulus by phosphorylation of a cytoplasmic interaction partner. Although plant receptor kinases represent the largest protein kinase family, only handfuls are well characterized. By sequence identity (orthology), a family of leucine-rich repeat receptor kinases from Arabidopsis thaliana was identified as AtORK11 (Arabidopsis thaliana Ovule Receptor Kinase 11). Based upon previous results from its ortholog in Solanum chacoense, the ovary- specific ScORK11 receptor kinase, we hypothesized that members of the AtORK11 receptors would be involved in gametophyte development and reproduction. In order to characterize the role of the four family members (AtORK11a, AtORK11b, AtORK11c and AtORK11d), a T-DNA insertional mutant strategy was undertaken, as well as bimolecular fluorescence complementation assays (BiFC). No precise function could be assigned to the double mutants although a dominant negative strategy revealed an involvement in gametophytic development. It was also shown that all of the receptors could form homodimers as well as heterodimers in a heterologous system, suggesting high functional redundancy for the AtORK11 family.

Au cours du développement des végétaux, de l’établissement de l’identité cellulaire des premiers organes au guidage du tube pollinique, la communication cellule à cellule est d’une importance capitale. En réponse, les voies de signalisation moléculaires sont élaborées pour la perception d’un signal extérieur et la transduction en une réponse génique via une cascade intracellulaire. Les récepteurs kinases font partie des protéines perceptrices des stimuli et constituent chez les plantes une catégorie de protéines avec une occurrence considérable, mais dont très peu d’informations détaillées sont disponibles à ce jour. Une famille de récepteurs kinases chez Arabidopsis thaliana, AtORK11 (Arabidopsis thaliana Ovule Receptor Kinase 11), a été identifiée par orthologie à un récepteur spécifique aux ovaires chez une solanacéee sauvage, Solanum chacoense. La fonction présumée de cette famille de récepteurs kinases de type leucine-rich repeat, suggérée par son patron d’expression, implique les événements relatifs au développement des gamétophytes et à la reproduction. Afin de caractériser la fonction des quatre gènes de la famille (AtORK11a, AtORK11b, AtORK11c et AtORK11d) une stratégie d’analyse de mutants d’insertion de l’ADN-T et d’évaluation du mode d’action par complémentation bimoléculaire par fluorescence (BiFC) a été entreprise. Aucune fonction précise n’a pu être attribuée aux doubles mutants d’insertion, par contre la surexpression d’une construction dominante négative indique un rôle dans le développement gamétophytique. Il a aussi été démontré que les quatre récepteurs peuvent interagir par homodimérisation aussi bien que par hétérodimérisation. Une hypothèse de redondance fonctionnelle est ainsi mise à jour parmi la famille des gènes AtORK11. / Cell to cell communication is paramount during plant developmental processes, from cellular identity in early organogenesis to pollen tube guidance. In response to this requirement, molecular cell signalling is used to perceive an external signal and transduce the response by an intracellular signalling cascade leading to specific gene activation. The sensing protein is typically a receptor kinase, which will transduce the stimulus by phosphorylation of a cytoplasmic interaction partner. Although plant receptor kinases represent the largest protein kinase family, only handfuls are well characterized. By sequence identity (orthology), a family of leucine-rich repeat receptor kinases from Arabidopsis thaliana was identified as AtORK11 (Arabidopsis thaliana Ovule Receptor Kinase 11). Based upon previous results from its ortholog in Solanum chacoense, the ovary- specific ScORK11 receptor kinase, we hypothesized that members of the AtORK11 receptors would be involved in gametophyte development and reproduction. In order to characterize the role of the four family members (AtORK11a, AtORK11b, AtORK11c and AtORK11d), a T-DNA insertional mutant strategy was undertaken, as well as bimolecular fluorescence complementation assays (BiFC). No precise function could be assigned to the double mutants although a dominant negative strategy revealed an involvement in gametophytic development. It was also shown that all of the receptors could form homodimers as well as heterodimers in a heterologous system, suggesting high functional redundancy for the AtORK11 family.

Cancer progression is a multi-step process requiring cells to acquire specific properties that aid the neoplastic growth. One such property is the ability to survive in the absence of matrix-attachment, a critical necessity for cells to traverse in circulation and seed metastases. Therefore, understanding the signalling mechanisms that protect cells from undergoing death in matrix-deprived condition, termed as anoikis, is important. We have used two systems to study this, one involving experimental transformation model, and another involving cancer cell lines. In the in vitro transformation model system involving the serial introduction of oncogenes, the ability to survive in anchorage-independent condition and generate spheres/colonies was dependent on the presence of the Simian Virus Small T antigen, SV40 ST. We identified that the viral antigen mediates its effects, at least in part, by activating the master metabolic regulator and cellular stress kinase AMP-activated protein kinase (AMPK) leading to maintenance of energy homeostasis. Consistent with this, our lab has previously identified both activation of AMPK upon matrix-deprivation in breast cells, as well as its requirement for survival under these conditions. However, a pathway often associated with survival under matrix-deprivation is the PI3K/Akt pathway. Surprisingly, we observed an AMPK-dependent decrease in Akt activity under conditions of matrix-detachment. Since this was contrary to the general notion, we probed deeper into a possible crosstalk between these two kinases. Our work revealed that AMPK activation in suspension inhibits Akt via upregulation of a known Akt phosphatase, pleckstrin homology domain leucinrich repeat protein phosphatise (PHLPP). We further show that the AMPK-PHLPP-Akt signalling axis is important for anoikis-resistance and metastasis. In addition, our results point to a yet unidentified protumorigenic role of PHLPP in breast cancer progression. With an aim to identify cellular proteins differentially regulated upon AMPK activation in breast cancer cells, we undertook a proteomics approach. Using 2-dimensional gel electrophoresis followed by mass spectrometric analysis, we identified some candidate proteins. We have validated the increase in levels of one of these proteins, annexin A2, in cancer cells upon AMPK activation. In summary, the present study unveils novel oncogenic functions of AMPK in cancer cells under the stress of matrix-deprivation. Furthermore, our results elucidate a double-negative feedback loop between two critical cellular kinases AMPK and Akt, and also identify a novel pro-tumorigenic role of PHLPP in breast cancer. In addition, we identify PHLPP and annexin A2 as novel proteins upregulated by AMPK in cancer cells. Thus, our results begin to identify pathways utilised by cancer cells to aid anchorage-independent growth, a critical step for cancer metastasis. Based on our results, inhibition of AMPK or perturbation of signalling axes involving AMPK, and PHLPP or annexin A2 might be considered as novel therapeutic approaches to combat cancer progression

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