Experiments were conducted to test for ricin content in tissue samples from four castor cultivars, developing castor seed, germinating castor seedlings, and chemically and heat treated seed meal. Ricin content of each sample was examined via Western blotting with ricin A-chain specific antibodies. Results indicate that ricin is present solely within castor endosperm and is not present any other tissues. Samples from developing seed and germinating seedlings indicate ricin production begins around day 28 post pollination, and ricin is absent from the seedling 6 days after the onset of radicle emergence. This would seem to indicate that the purpose of ricin is to protect the seed and not the entire plant. Ricin content of seed meal treated separately with heat and chemicals was tested. It was found that hot-pressing of the seed was sufficient to denature ricin in the seed meal.

Doctor of Philosophy / Department of Biology / Jyoti Shah / Localized inoculation of a plant with an avirulent pathogen results in the activation of systemic acquired resistance (SAR), a defense mechanism that confers enhanced resistance against a variety of pathogens. The activation of SAR requires the translocation of an unknown signal from the pathogen-inoculated organ to the other organs where defenses are primed to respond faster in response to a future attack by a pathogen. Previous studies with the Arabidopsis thaliana dir1 (defective in induced resistance1) and sfd1 (suppressor of fatty acid desaturase deficiency1) mutants implicated a role for plant lipids in the activation of SAR. DIR1 encodes a putative lipid transfer protein and SFD1 encodes a dihydroxyacetone phosphate (DHAP) reductase involved in plastid glycerolipid metabolism. To further evaluate the role of DHAP reductases and plastid lipids in SAR, the involvement of two additional putative DHAP reductase encoding genes (AtGPDHp and AtGPDHc) and the SFD2 gene, which like SFD1 is involved in plastid glycerolipid metabolism, in SAR was evaluated. Only SFD2 was found to be essential for SAR. Although the lipid profile of the sfd2 mutant was similar to that of the fad5 (fatty acid desaturase 5) mutant, sfd2 is not allelic with fad5 and does not influence FAD5 expression. The SFD2 gene was mapped to an 85 kilo basepairs (kb) region on the third chromosome of Arabidopsis. The lipid composition defect of the sfd2 mutant was partially complemented by two independent recombinant bacterial artificial chromosomes (BACs) that contained genomic DNA spanning the wild type SFD2 locus. The role of plastid synthesized glycerolipids in the activation of SAR was further evaluated by characterizing SAR in additional Arabidopsis mutants that were deficient in plastid lipid metabolism. The requirement of MGD1 (MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE 1), DGD1 (DIGALACTOSYL-DIACYLGLYCEROL SYNTHASE 1) and FAD7 (FATTY ACID DESATURASE 7) genes in SAR, confirmed the essential role of plastid glycerolipids, presumably a galactolipid-dependent factor, in signaling associated with the SAR.

The Arabidopsis SON1 F-box protein was implicated in regulating a pathogen defense pathway, but its exact function in wild-type plants is unknown. As an F-box protein it was predicted that SON1 would assembles into a SON1-SCF ubiquitin ligase complex that recruits specific plant defense-related proteins for proteolysis. The yeast 2-hybrid assay was used to screen for potential substrates for a putative SON1-SCF ligase, leading to the identification of Arabidopsis LNK2 as a SON1-binding factor. Comprehensive protein-protein interaction analysis has shown that the binding of SON1 to LNK2 protein is specific, because closely related, full-length Arabidopsis F-box proteins do not interact with LNK2. However, when tested in isolation, some fragments derived from the paralog proteins did bind SON1, suggesting that higher order structure or inter-domain interference affects the ability of SON1 to recruit substrate. When analyzed for interaction domains, three regions of SON1 were identified that bind to LNK2 and a LNK2 binding region spans a conserved amino acid region. Phylogenetic analysis revealed that there is a paralogous gene called LNK1 in Arabidopsis, and both LNK1 and LNK2 are restricted to the plant kingdom. LNK2 and LNK1 are seen to be widely distributed in embryophyte seed and spore plants. Genetic analysis and complementation tests showed that LNK1 and LNK2 regulate flowering and photo-morphogenesis redundantly. Though lnk1 and lnk2 mutants look similar to wild-type plants, lnk1 lnk2 double mutant plants possess a long hypocotyls and flower late compared to wild-type plants. Because SON1 is implicated in plant defense, lnk mutants were assessed for susceptibility to a virulent oomycete pathogen, Hyaloperonospora arabidopsidis (Hpa). Interestingly, lnk mutants supported less disease development, suggesting a role of the wild-type LNK proteins in the enabling of pathogen colonization or in repressing host defenses. To confirm that each of the phenotypes described were a consequence of lnk1 and lnk2 mutations, wild type LNK1 and LNK2 were introduced into lnk1 lnk2 plants and examined. For most phenotypes, genetic complementation and thus restoration of a WT phenotype was observed. However, differences were uncovered in the ability of LNK genes to rescue the phenotypes, indicating specialization of function. The interaction of LNK2 with SON1 suggests that SON1-dependent ubiquitination and proteasomal degradation may control LNK2 abundance. I show by a cell free protein degradation assays that proteasome-based degradation of LNK2 as well as LNK1 is possible. Data showed that SON1 binds to ASK1 in-planta suggest the existence an SCFSON1 complex that targets LNK2 for polyubiquitination and its subsequent degradation by the proteasome. The data presented in this dissertation indicates a role for LNKs in flowering and plant defense and also suggest that proteasome-base regulation of LNK turnover may be utilized to regulate LNK protein abundance and proper maintenance of the circadian clock.

Ratoon stunting disease (RSD) is a serious disease that affects all sugarcane producing countries. The major symptom of RSD is plant growth reduction, which is only seen in ratoon plants, causing up to 80% biomass reduction depending on environmental conditions. The disease is due to Leifsonia xyli subsp. xyli (Lxx), a gram-positive and nutritionally fastidious bacterium that so far has been found to specifically colonize the xylem vessels of sugarcane. However, the successful early detection of this pathogen is currently the main challenge for RSD prevention. Breeding for resistance to RSD, although not in practice, is a viable control measure. Since sugarcane varieties differ in relation to their degree of colonization by Lxx and losses are directly related to population densities of the pathogen in the plant, a promising breeding strategy would be to select for genotypes that are resistant to bacterial multiplication. Thus, knowledge on the responses of sugarcane to RSD at the \"omics\" level is an essential starting step to identify key metabolic targets for breeding resistant varieties. The overall goal of this study is to determine the metabolic profiles of a susceptible (CB49-260) and resistant (SP80-3280) variety inoculated or not with Lxx and to compare the results with existing proteomic and transcriptomic data to define a core of targets (proteins, genes, and metabolites) that can be tested as markers of resistance in a collection of sugarcane varieties. Bacterial titers were quantified by Real-Time PCR (qPCR). The metabolites were profiled from the leaves and from the xylem saps collected at 30 and 120 days after inoculation (DAI). Untargeted analysis were performed with Gas Chromatography - Mass Spectrometry (GC-MS) and were carried out on leaves and sap from 120 DAI. Targeted analysis was executed with Liquid Chromatography - Tandem Mass Spectrometry (LC-MS/MS) on both tissues at both timepoints. To validate metabolomics results, a set of metabolites was chosen to be tested in vitro, in order to detect growth alterations caused to Lxx. qPCR confirmed the susceptibility of CB49-260 as it had higher titers than SP80-3280. Global analysis revealed that both varieties and tissues have different metabolic profiles but that those differences are more quantitative than qualitative. The targeted approach identified more amino acids, sugars, organic acids and phosphorylated compounds in the non-inoculated susceptible genotype, while the resistant one had higher abundance of phenolics. It was also shown that inoculation with Lxx results in more relative abundance of amino acids, organic acids, phosphorylated compounds and phenolics. Furthermore, a key amino acid for Lxx survival was related to inoculation on both varieties, as well as a known phenolic compound related to plant defense. Distinguished phenolics resulting from the targeted analysis were selected to evaluate their effect on Lxx growth in vitro. Although some compounds caused inhibition, further optimization of the methodology is needed to confirm these results. / O Raquitismo-da-soqueira (RSD) é uma grave doença que afeta todos os paises produtores de cana-de-açúcar. O principal sintoma do RSD é tamanho reduzido das plantas, observado apenas nas plantas-soca, o que pode resultar em perdas de biomassa em até 80%, dependendo das condições climáticas. A doença é causada por Leifsonia xyli subsp. xyli (Lxx), uma bactéria gram-positiva e fastidiosa, descrita até o presente momento como hospedeira natural apenas da cana-de-açúcar, colonizando principalmente os vasos do xilema. Todavia, a detecção precoce deste patógeno é o principal desafio para prevenção do RSD. O melhoramento genético para resistência ao RSD, apesar de viável, não é uma medida de controle adotada na prática. Como existe diferenças entre as variedades de cana em relação ao grau de colonização por Lxx e as perdas estão diretamente relacionadas ao título bacteriano, uma estratégia de melhoramento promissora é a seleção de genótipos que apresentam resistência à multiplicação bacteriana. Portanto, o conhecimento das respostas da cana-de-açúcar ao RSD em termos \"ômicos\" é um passo inicial primordial para a identificação de alvos-chave para melhorar variedades resistentes. O objetivo geral deste estudo foi determinar os perfis metabólicos de duas variedade, uma suscetível (CB49-260) e uma resistente (SP80-3280) inoculada ou não com Lxx e comparar os resultados com dados já existentes de proteômica e transcriptômica para definir um núcleo de alvos (proteínas, genes e metabólitos) que possam ser testados como marcadores de resistência em uma coleção de cana-de-açúcar. Os títulos bacterianos foram quantificados por PCR em tempo real (qPCR). Os perfis metabólicos foram elaborados a partir de folhas e fluído xilemático coletados aos 30 e 120 dias após inoculação (DAI). A análise não-direcionada foi realizada por cromatografia gasosa acoplada à espectrometria de massas (GC-MS), usando folhas e extratos coletados aos 120 DAI. Já a análise direcionada foi efetivada via cromatografia líquida acoplada à espectrometria de massas em tandem (LC-MS/MS), em ambos tecidos e tempos de coleta. Para validar os resultados de metabolômica, um grupo de metabólitos destacado nas análises de metabolômica foi escolhido para testes in vitro e por fim detectar alterações no crescimento de Lxx. O resultado do qPCR confirmou a suscetibilidade da CB49-260, pois esta continha títulos superiores à SP80-3280. A análise global revelou que ambos variedades e tecidos possuem perfis metabólicos distintos, porém essas diferenças foram mais quantitativas que qualitativas. A análise direcionada identificou mais aminoácidos, açúcares, ácidos organicos e compostos fosforilados no genótipo suscetível não-inoculado, enquanto que o resistente apresentou maior abundância de compostos fenólicos. Também foi demonstrado que a inoculação com Lxx resultou em maior quantidade de aminoácidos, ácidos orgânicos, compostos fosforilados e fenólicos. Ademais, um aminoácido essencial à sobrevivência de Lxx foi relacionado à inoculação de ambas variedades, assim como um composto fenólico relacionado a defesa de plantas. O teste in vitro mostrou que, apesar de alguns compostos causarem inibição, é necessário aprimorar a metodologia utilizada para confirmar os resultados obtidos.

Os ácaros Tetranychus evansi e Tetranychus urticae são pragas conhecidas de tomateiros. O ácaro T. urticae induz as defesas diretas e indiretas das plantas de tomate. Já a espécie T. evansi suprime as defesas diretas e indiretas e protege seu sitio de alimentação com a produção de teia densa. Não se conhece o desempenho de insetos pragas em plantas infestadas por ácaros T. evansi. O objetivo deste trabalho foi avaliar, em campo, a colonização de plantas por outros artrópodes após serem infestadas por T. evansi ou por T. urticae. No primeiro capítulo da tese foram feitos experimentos em campo para avaliar o desempenho de fêmeas de T. evansi em plantas de tomate limpas (controle) ou infestadas por T. evansi ou infestadas por T. urticae. Foi observado que a oviposição de fêmeas de T. evansi em plantas infestadas anteriormente por T. urticae foi significativamente menor do que observada em plantas limpas ou infestadas antes por T. evansi. Também foi avaliada a preferência de artrópodes em cada tratamento. As plantas foram separadas em três tratamentos (planta limpa, infestada com 400 ácaros T. urticae ou T. evansi) e após 4 dias foram posicionadas em um hexágono, em campo. A maior quantidade de herbívoros foi encontrada em plantas limpas do que em plantas infestadas com T. urticae ou com T. evansi. Dentre os herbívoros com maior abundância e frequência estavam as moscas brancas Bemisia tabaci (Biotipo B). No segundo capítulo foi estudada a preferência das moscas brancas (B. tabaci) em plantas limpas ou infestadas por T. evansi ou T. urticae com e sem teia. As moscas brancas foram encontradas em maior quantidade em plantas limpas ou infestadas por T. urticae. No terceiro capítulo foi avaliada a oviposição e desempenho de moscas brancas B. tabaci em plantas limpas ou infestadas com ácaros T. evansi ou T. urticae. As moscas brancas ovipositaram mais em plantas infestadas por T. evansi e em plantas limpas. Não houve diferença significativa na taxa de mortalidade de ninfas de moscas brancas entre os tratamentos. Nessa tese foi comprovado que artrópodes em campo evitam plantas infestadas por T. evansi. / The spider mites Tetranychus evansi and Tetranychus urticae are pests of tomato plants. The species T. urticae induces direct and indirect defenses of tomato plants. The species T. evansi suppresses direct defenses of tomato plants and protects its feeding site with dense web. The performance of insect pests on plants infested by T. evansi mites is not known. The objective of this study was to evaluate the colonization of plants infested by T. evansi and T. urticae by other arthropods in the field. In the first chapter of this thesis I evaluated the performance of females of T. evansi in clean or infested tomato plants by T. evansi or T. urticae. I observed that the oviposition of T. evansi females was significantly lower on plants previously infested by T. urticae than on clean or infested plants by T. evansi. In another experiment, I evaluated the preference of arthropods in each treatment. Plants were divided into three treatments (clean plant, infested with 400 T. urticae or 400 T. evansi) and after 4 days they were positioned in a hexagon in the field. The largest amount of herbivores were found on clean plants, than on plants infested by T. urticae or T. evansi. The whitefly Bemisia tabaci (byotipe B) was the most abundant herbivore found on the plants. In the second chapter, I studied the preference of whitefly (B. tabaci) for clean plants or plants infested by T. evansi or T. urticae with and without web. The whiteflies were found in greater quantities in clean plants or plants infested by T. urticae. In the third chapter, I assessed the oviposition and performance of whitefly B. tabaci in clean plants or infested plants with T. evansi or T. urticae. The whiteflies laid more eggs on plants infested by T. evansi and clean plants. There was no significant difference in mortality of whiteflies nynphs between treatments. This thesis has been proved that arthropods in the field avoid plants infested by T. evansi, in the field.

Salicylic Acid Binding Protein2 (SABP2) is an enzyme known to play important role in SA mediated pathway. SBIP-428 (SABP2 Interacting Protein-428), a SIR2 like deacetylase, has been found to interact with SABP2. We demonstrate that SBIP-428 functions as a Sirtuin deacetylase. We show that SBIP-428 itself is lysine acetylated. Interactions of a SBIP-428 with SABP2 also raised the possibility of SABP2 itself being lysine acetylated. The recombinant purified SABP2 or native partially purified SABP2 displayed no acetylation. In response to TMV infection, the expression of SBIP-428 was down regulated at 48 hpi. In addition, SBIP-428 was up regulated in plant known to accumulate less SA. Taken together expression of SBIP-428 is negatively correlated to the levels of SA in plants. The AtSRT2 plants exhibit no altered growth phenotype but exhibit a higher pathogen resistance against bacterial pathogen. Our results indicate that SBIP-428 is an important regulator in plant defense pathway.

Abscisic acid (ABA) induces RD22, responsive to dehydration stress gene. Salicylic acid (SA) has been the focus of research due to its role against pathogens and abiotic stress. Interaction between ABA and SA signaling pathways is still poorly understood. SA-Binding Protein 2 (SABP2) converts methyl salicylate to SA. An attempt was made to identify proteins that interact with SABP2 using a yeast 2-hybrid screening. Several interactors were identified. One of them, SA-Binding Protein 2 Interacting Protein-355 (SBIP-355), showed high homology to RD22. Bioinformatic approaches showed that SBIP-355 contains a BURP domain. Phylogenetic analysis reveals that SBIP-355 clustered into the clade of RD22-like proteins. Thus, SBIP-355 gene might be a stress-inducible gene and encodes a dehydration-responsive protein, which is important for the stress tolerance of tobacco. The complementary DNA (cDNA) of tobacco SBIP-355 was cloned into pDEST-17 vector and then expressed in E. coli to detect the expression of SBIP-355 protein.

Plants are in a perpetual evolutionary arms race with a wide range of pathogens. Their sessile nature has led plants to evolve defense mechanisms that can quickly recognize a unique stressor and deploy a resistance tailored for a specific attack. The salicylic acid (SA) mediated defense pathway has been shown to be one of the major defense tactics plants can initiate to defend themselves against microbial pathogens. Following a pathogen attack high levels of methyl salicylate (MeSA) are produced that can be converted to SA by the enzyme salicylic acid binding protein 2 (SABP2). A yeast two-hybrid screening was performed to identify protein interactions with SABP2 to better understand the regulation of the enzyme on a cellular level. SBIP-436 is an interacting protein of tobacco SABP2 which showed high homology to phospholipase D-δ (PLD- δ). With an abundance of stimulators PLD- δ may be a lipid signaling modulator developed to perform various functions in different situations. PLD- δ may be a key player in a lipid signaling cascade in the SA mediated defense pathway. We present a novel Nicotiana tabacum PLD- δ putative gene construct. We demonstrate that the putative PLD- δ is subject to alternative splicing and its expression is differentially modulated under biotic and abiotic stress. Our results indicate that this putative PLD- δ may play a role in the SA mediated defense pathway.

Expanding upon our understanding of plant defense is critical, particularly with the perilous threats of climate change and overpopulation to our food security, health and well-being. In this study, we focused on plant defense using two distinct approaches. First, we performed a proteomic analysis of plant exosome-like nanoparticles in order to elucidate their defense related protein cargo. Secondly, we used a wheat antimicrobial protein, puroindoline, as a fusion partner for the expression of recombinant proteins in rice endosperm. Plant exosome-like nanoparticles (ELP) were isolated from fresh tomato and subjected to mass spectrometry (MS) analysis. The ELPs were compared to fresh pressed tomato juice, and the proteins that were significantly upregulated in the ELPs were analyzed for their defensive properties. Bioinformatic analysis identified 30 proteins upregulated in the ELPs, with a majority of these being involved in plant defense. Puroindoline is a protein found in soft wheat varieties. A unique feature of this protein is the presence of a tryptophan-rich domain, which causes it to localize and tether onto starch granule surfaces; a property we are seeking to exploit for recombinant protein isolation. We hypothesized that when expressed in a pin-null crop, such as rice, puroindoline along with its fusion partner will localize and adhere to starch granule surfaces. PIN fusions were expressed in rice, and their subcellular localization was determined by immunolocalization. It was observed that PIN localizes to rice starch ii granules in vitro and in planta, and retains its starch granule binding abilities as a fusion partner. To identify other possible starch granule binding fusion partners, an anhydrous cleavage method was developed that can scan dry biological materials for associated proteins, in this case the starch granule surface. Incubation of our cleavage reagent with isolated rice starch granules yielded several cleavage products as determined through SDS-PAGE. These cleavage products were compared with previous proteomic data of trypsin digested rice starch granules.

Systemic acquired resistance (SAR) is a type of plant defense mechanism that is induced after a localized infection and confers broad-spectrum immunity against related or unrelated pathogens. During SAR, a number of chemical signals and proteins generated at the site of primary infection travel to the uninfected tissues and are thought to alert the distal sites against secondary infections. Glycerol-3-phosphate (G3P) is one of the chemical signals that play an important role in SAR. G3P is synthesized in the cytosol and chloroplasts via the enzymatic activities of G3P Dehydrogenase (G3Pdh) or Glycerol Kinase (GK). Interestingly, a mutation in three of the five G3Pdh isoforms or GK impairs SAR by lowering the pathogen induced G3P pool. This suggests that total cellular pool of G3P is critical for SAR. To determine factors contributing to G3P flux between various subcellular compartments I analyzed the role of putative G3P transporters in G3P flux and SAR. The Arabidopsis genome encodes five isoforms of G3P Permeases (G3Pp) and these transmembrane proteins are predicted to localize to plasma membrane, chloroplast or mitochondria. At least two G3Pp isoforms (G3Pp1 and G3Pp3) were able to complement the Escherichia coli mutant impaired in the uptake of G3P into the cytoplasm. Characterization of Arabidopsis G3Pp mutants showed that a mutation in G3Pp2, G3Pp3 and G3Pp4 compromised SAR but not local resistance. Furthermore, this SAR defect could only be complemented by exogenous application of G3P. The G3Pp mutants accumulated wild-type-like levels of G3P suggesting that the subcellular compartmentalization of G3P might contribute to the induction of SAR.