Les champignons sont les principaux agents pathogènes des plantes. Leur étude est donc essentielle pour contrôler les maladies et maintenir un bon rendement de production agricole. La nutrition de ces pathogènes est basée sur l'absorption de nutriments, préalablement dégradés par un arsenal d'enzymes lytiques secrétées. La sécrétion des protéines est assurée par le trafic intracellulaire mettant en jeu de nombreuses vésicules. Chez les champignons filamenteux, ces vésicules ont été visualisées en microscopie électronique mais le processus mis en jeu pour leur biogénèse n'est toujours pas élucidé. L'identification de ce mécanisme est un donc un prérequis pour comprendre la sécrétion de facteurs de virulence. Dans ce but, un mutant non pathogène altéré au niveau de l'expression du gène codant la chaine lourde de la clathrine a été sélectionné parmi une banque de mutants générés chez le champignon nécrotrophe Botrytis cinerea. Le gène codant pour la chaine lourde de la clathrine est essentiel chez de nombreux organismes, ainsi un mutant dominant négatif de la chaine lourde de la clathrine a été généré et confirme la perte de pathogénicité. La caractérisation du mutant par une approche de protéomique a mis en évidence un défaut de sécrétion de 82 protéines incluant des facteurs de virulence connus. Un défaut de production de vésicules intracellulaires a également été constaté. Par ailleurs, le marquage de la clathrine à la GFP a permis de préciser sa localisation dans les cellules fongiques. Enfin, de façon surprenante, aucun défaut d'endocytose n'a été constaté au sein des mutants déficients en clathrine. Cette étude met en évidence pour la première fois le rôle essentiel de la clathrine dans le processus infectieux d'un champignon pathogène ainsi que son rôle dans a sécrétion de facteurs de virulence / Fungi are the most important plant pathogens on agricultural and horticultural crops. Study of fungal pathogens remains essential to understand pathogenic process and control plant diseases. These organisms secrete high amount of degrading enzymes involved in plant decomposition and they feed by absorption of degraded nutriments. Secretory proteins were described to be transported form Endoplasmic Reticulum and Golgi apparatus to extracellular space through intracellular vesicles. In filamentous fungi, intracellular vesicles were observed using electron microscopy but their biogenesis process is still unknown. Therefore, elucidation of the process and the identification of proteins involved in secretory vesicles biogenesis remains a challenge to understand virulence factors delivery. A nonpathogenic mutant altered in the expression of the gene coding for clathrin heavy chain was selected in a random mutant library generated in the necrotrophic pathogen Botrytis cinerea,. This gene is essential in many organisms, thus a clathrin dominant negative mutant was generated and confirming the nonpathogenic phenotype observed on several host plant. In eukaryotic cells, clathrin heavy chain is mainly described to be involved in endocytosis, but it is also essential for high density secretory vesicles formation in yeast. Characterization of the mutants using a proteomic approach revealed a secretion defect of 82 proteins including known virulence factors, as Plant Cell Wall Degrading Enzymes and elicitors. Furthermore, the clathrin mutant revealed a strong reduction of intracellular vesicles production. Clathrin was also localized in living cells using fluorescent GFP-tag protein. Endocytosis was also studied and surprisingly, any observable defect was observed for clathrin mutants. This study demonstrated for the first time the essential role of clathrin in the infectious process of a fungal pathogen and its role in virulence factors secretion

Austropuccinia psidii (sin Puccinia psidii) é um fungo biotrófico que infecta diversos gêneros de mirtáceas. É uma ferrugem (Puccinialles) nativa da América do Sul que apresenta ampla distribuição e rápida dispersão geográfica, alcançando atualmente a Austrália, centro de diversidade das mirtáceas. Este patógeno tem alarmado a comunidade científica devido à vasta capacidade de dispersão e por causar perdas econômicas consideráveis em culturas de interesse comercial, com destaque na eucaliptocultura. Apesar de sua importância, estudos relacionados ao patossistema A. psidii - Eucalyptus ainda são incipientes, sendo que o entendimento das bases moleculares envolvidas na interação planta-patógeno é essencial para adoção de medidas de controle eficazes e duradouras. Atualmente, a busca por candidatos a efetores tem crescido consideravelmente, pois são moléculas capazes de alterar a fisiologia do hospedeiro, suprimindo ou ativando os mecanismos de defesa. Dessa forma, o objetivo do presente estudo foi identificar in silico genes candidatos a efetores e validá-los por meio de análise de expressão por RT-qPCR. Para predição dos genes candidatos a efetores foi utilizado o genoma parcial de A. psidii e uma pipeline específica, baseada em diversos parâmetros, tais como: presença do peptídeo sinal, ausência de domínio transmembrana e superfície de ancoragem e pequeno tamanho. Foram identificados 2.886 candidatos, dos quais 13% apresentaram similaridade com Puccinialles. As categorias mais importantes de localização subcelular preditas foram apoplasto (87,3%), cloroplasto (7%) e núcleo (4,1%). Oito candidatos foram selecionados para análise de expressão após mineração em dados de RNA-seq. Os ensaios foram realizados in vitro com uredósporos de duas populações distintas de A. psidii, uma proveniente de eucalipto (MF-1) e outra de S. jambos (GM-J1) e as amostras foram coletadas em 6, 12 e 24 horas após inoculação (h.a.i). O perfil de expressão dos candidatos variou entre os tempos investigados, sendo que dois candidatos mostraram altos valores de expressão em todos os tempos, ao passo que quatro foram mais expressos nos tempos iniciais (6 e 12 h.a.i). Os tempos iniciais correspondem às fases de germinação e pré-penetração, fortalecendo a predição desses quatro candidatos como apoplásticos. Foi observado um perfil diferencial de expressão entre as duas populações do patógeno, o que sugere uma provável modulação pelo hospedeiro de origem na expressão dos candidatos a efetores, fato ainda pouco abordado na literatura e que deve ser melhor investigado. Embora o trabalho tenha sido realizado in vitro, foi possível selecionar potenciais candidatos para continuidade dos estudos, incluindo a validação in planta e caracterização funcional. Os dados são relevantes em vista a escassez de informações biológicas desse patossistema e fornecem uma visão inicial de como esses efetores atuam na interação planta-patógeno. / Austropuccinia psidii (sin Puccinia psidii) is a biotrophic fungus that infects several genera of Myrtaceae. It is a rust (Puccinialles) native from South America that displays a broad distribution after have shown a quick geographic dispersion, being currently present even in Australia, which is Myrtaceae\'s diversity center. This pathogen has alarmed the scientific community due to its wide dispersion ability and because of considerable economic losses occurring in crops of commercial interest, especially eucalyptus cultures. Despite its importance, studies related to A. psidii-Eucalyptus pathosystem are still incipient and the understanding of the molecular basis involved in the plant-pathogen interaction is essential to develop effective and durable control mechanisms. Currently, the search for effector candidates has increased considerably since they are molecules that alter the host physiology, suppressing or activating the defense mechanisms. In this study, we aimed to identify in silico effectors candidate genes and also to validate them by expression analysis (RT-qPCR). To predict the effector candidate genes, we used the partial genome of A. psidii and a specific pipeline based on several parameters, such as small size, presence of signal peptide, absence of transmembrane domain and anchorage surface. A total of 2,886 candidates were identified, from which 13% are similar to Puccinialles. The most important categories of predicted subcellular localization were apoplast (87,3%), chloroplast (7%) e nucleus (4,1%). Eight candidates were selected for expression analysis after mining in RNA-seq data. The in vitro assays were carried out with uredospores from two distinct populations of A. psidii: one from eucalyptus (MF-1) and other from S. jambos (GM-J1); the samples were collected 6, 12 and 24 hours after inoculation (h.a.i). The expression profile of the candidates was distinct among the times investigated. The results showed that two candidates had high expression values in all times evaluated, while four were more expressed at 6 and 12 h.a.i. These times correspond to the germination and pre-penetration phases, corroborating with the prediction of these four candidates as apoplastics. A differential expression profile was found between the two pathogen populations, suggesting a probable modulation by the origin host in the expression of effectors candidates, what has not been extensively investigated and discussed in the literature on the topic. Although this study has been performed in vitro, it was possible to select potential candidates for further investigations, including in plant validation and functional characterization. The data presented here are relevant considering the scarcity of biological information about this pathosystem, besides providing an initial insight on how these effectors act in the plant-pathogen interaction.

Turnip crinkle virus (TCV) has been implicated in the suppression of the hypersensitive response (HR), a type of programmed cell death induced during active resistance in Arabidopsis thaliana. In order to investigate the involvement of individual viral components in mediating suppression, TCV genes were cloned for use in an Agrobacterium tumefaciens mediated transient expression in Nicotiana benthamiana. Agroinfiltration of the HR-inducing avrPto/Pto system in conjunction with individual TCV genes has identified the p38 gene, which encodes the viral coat protein, as the gene responsible for the cell death suppression phenotype. The extent of cell death suppression by coat protein was quantified and found to be equal to the level of suppression by the whole virus and AvrPtoB, another cell death inhibitor from bacteria. Thus, the coat protein alone is sufficient to inhibit the HR in plants. Further, the effect of TCV on HR initiation by an avirulence factor from an unrelated bacterial pathogen was investigated. The presence of TCV does not affect the production, secretion or cellular processing of the bacterial avirulence factor.

Upon detection of a pathogen, plants initiate specific signaling events designed to prevent host colonization and pathogen proliferation. Appearance of the hypersensitive response (HR), a type of programmed cell death signifies activation of active defenses in response to a one-to-one recognition of host, Resistance or R gene, and pathogen, avirulence or avr gene, encoded products. Turnip crinkle virus (TCV), however, has been shown to suppress the ability of Col-0 Arabidopsis thaliana plants to produce the HR in response to an avirulence factor. The extent of suppression was quantified by measuring cellular electrolyte leakage resulting from programmed cell death. Interestingly, cellular ion leakage levels were significantly lower in TCV-infected plants when challenged with bacteria expressing either of two bacterial effectors avrRpt2 or avrRpm1, suggesting that TCV can suppress the HR to a range of HR-inducing avirulence factors. In order to determine the viral component(s) responsible for mediating this suppression, each of the five TCV open reading frames (ORFs) was tested using an Agrobacterium tumefaciens-mediated transient expression assay in Nicotiana benthamiana. Though sequencing of the five TCV clones revealed mutations in the p28, p88, and p9 clones, Agro infiltration of an HR-inducing system in conjunction with individual TCV ORFs, or combinations of, was used to gather data to determine the role each may possess in the suppression phenotype. Full-length TCV was also expressed in the presence of AvrPto/Pto to establish suppression phenotype in Nicotiana. To assay for suppression of cell death in a heterologous system, both the mutant and wild-type clones were also tested in yeast for cell-death suppression induced by hydrogen peroxide exposure.

Shuk-man Chow. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 171-186). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgement --- p.viii / General abbreviations --- p.x / Abbreviations of chemicals --- p.xi / List of figures --- p.xii / List of Tables --- p.xiii / Table of contents --- p.xv / Chapter 1. --- Literature review / Chapter 1.1. --- General introduction to rice disease --- p.1 / Chapter 1.1.1. --- Pathogenesis of Bacterial Leaf Blight (BLB) --- p.1 / Chapter 1.1.2. --- Pathogenesis of rice blast --- p.2 / Chapter 1.1.3. --- Control of rice diseases --- p.3 / Chapter 1.2. --- Plant defense mechanisms --- p.4 / Chapter 1.2.1. --- Basal resistance in plants --- p.4 / Chapter 1.2.2. --- Wound induced defense response --- p.5 / Chapter 1.2.3. --- Pathogen induced host defense response --- p.6 / Chapter 1.3. --- Structure of R gene products --- p.7 / Chapter 1.4. --- Recognition between R and Avr proteins in rice --- p.8 / Chapter 1.5 --- Current knowledge on Xa resistance and AvrXa avirulence protein --- p.9 / Chapter 1.6 --- Current knowledge on Pi resistance and AvrPi avirulence protein --- p.10 / Chapter 1.7 --- Pathogen induced signal transduction cascade --- p.12 / Chapter 1.7.1. --- R gene mediated signal transduction cascade --- p.12 / Chapter 1.7.2. --- Signal events of G-protein activation --- p.12 / Chapter 1.7.3. --- Signaling events for the accumulation of Ca2+ in cytosol --- p.13 / Chapter 1.7.4. --- Signaling events for oxidative burst --- p.14 / Chapter 1.7.5. --- MAPK cascade in defense signaling --- p.15 / Chapter 1.7.6. --- Transcriptional regulation of disease resistance related genes --- p.16 / Chapter 1.7.7. --- Translational regulation of disease resistance related genes --- p.17 / Chapter 1.8. --- Defense responses and defense related genes --- p.19 / Chapter 1.8.1. --- Pathogenesis related (PR) proteins --- p.20 / Chapter 1.8.2. --- Phytoalexins --- p.21 / Chapter 1.9. --- Disease resistance related genes common between rice blast and BLB resistance --- p.22 / Chapter 1.10. --- SA induced signal transduction pathway in rice --- p.23 / Chapter 1.11. --- Important tools facilitating the identification of disease resistance related genes from BLB resistant rice lines --- p.24 / Chapter 1.12. --- Hypothesis --- p.26 / Chapter 1.13. --- Project objective --- p.26 / Chapter 2. --- Materials and Methods --- p.27 / Chapter 2.1. --- Plant Materials --- p.27 / Chapter 2.2. --- Pathogen Inoculation --- p.27 / Chapter 2.3. --- RNA extraction --- p.29 / Chapter 2.4. --- Denaturing gel electrophoresis --- p.29 / Chapter 2.5. --- Subtraction libraries construction --- p.30 / Chapter 2.5.1. --- Cloning of disease resistance related genes --- p.32 / Chapter 2.5.1.1. --- pBluescript II KS (+) T-vector preparation --- p.32 / Chapter 2.5.1.2. --- Ligation --- p.32 / Chapter 2.5.1.3. --- Transformation --- p.32 / Chapter 2.5.1.4. --- Colony picking --- p.33 / Chapter 2.5.1.5. --- PCR amplification of DNA inserts --- p.33 / Chapter 2.5.1.6. --- Purification of PCR products --- p.34 / Chapter 2.6. --- Gene chips printing --- p.34 / Chapter 2.7. --- Probes synthesis and gene chips hybridization --- p.35 / Chapter 2.8. --- Standard-RNAs synthesis --- p.35 / Chapter 2.9. --- Data collection and analysis --- p.36 / Chapter 2.10. --- Sequencing --- p.36 / Chapter 2.11. --- cDNA synthesis --- p.37 / Chapter 2.12. --- RT-PCR --- p.38 / Chapter 2.13. --- DNA gel electrophoresis --- p.39 / Chapter 3. --- Results --- p.58 / Chapter 3.1. --- Construction of BLB gene chips --- p.58 / Chapter 3.1.1. --- Preparation of cDNA clones for gene chips construction --- p.58 / Chapter 3.1.2. --- Purification of PCR products on microtiter plate --- p.59 / Chapter 3.1.3. --- Gene chips construction --- p.59 / Chapter 3.1.4. --- DNA immobilization --- p.62 / Chapter 3.1.5. --- Probe synthesis --- p.62 / Chapter 3.1.6. --- Gene chip analysis --- p.65 / Chapter 3.1.6.1. --- Scanning --- p.65 / Chapter 3.1.6.2. --- Data analysis --- p.65 / Chapter 3.2. --- "Identification of disease resistance related genes commonly regulated by Xa2, Xal2 and Xal4 BLB resistance loci" --- p.70 / Chapter 3.2.1. --- "Signal perception, transduction and regulatory elements" --- p.71 / Chapter 3.2.1.1. --- Proteins involved in reversible phosphorylation cascade --- p.71 / Chapter 3.2.1.2. --- Proteins potentiate signal transduction through specific protein-protein interaction --- p.72 / Chapter 3.2.1.3. --- Other signal transduction components --- p.73 / Chapter 3.2.2. --- Transcriptional and translational regulatory elements --- p.74 / Chapter 3.2.2.1. --- Proteins involved in transcriptional regulation --- p.74 / Chapter 3.2.2.2. --- Proteins involved in post-transcriptional regulation --- p.75 / Chapter 3.2.2.3. --- Proteins involved in translational regulation --- p.76 / Chapter 3.2.3. --- "Oxidative burst, stress, apoptotic related genes" --- p.77 / Chapter 3.2.3.1. --- Stress related proteins --- p.77 / Chapter 3.2.3.2. --- Proteins involved in induction of oxidative burst --- p.78 / Chapter 3.2.3.3. --- PR proteins --- p.79 / Chapter 3.2.3.4. --- Proteolysis related proteins --- p.79 / Chapter 3.2.4. --- Cell maintenance and metabolic genes --- p.80 / Chapter 3.2.4.1. --- Antioxidant --- p.80 / Chapter 3.2.4.2. --- Metabolic genes --- p.81 / Chapter 3.2.4.3. --- Molecular chaperone --- p.82 / Chapter 3.2.4.4. --- Cell cycle regulators --- p.82 / Chapter 3.2.4.5. --- Cell wall maintenance --- p.83 / Chapter 3.2.4.6. --- Proteins involved in protein transport --- p.83 / Chapter 3.2.5. --- Unclassified/others --- p.84 / Chapter 3.3. --- Expression analysis of disease resistance related genes --- p.88 / Chapter 4. --- Discussion --- p.141 / Chapter 4.1. --- Differential expression of disease resistance candidates --- p.141 / Chapter 4.2. --- Disease resistance signal transduction components --- p.143 / Chapter 4.2.1. --- Reversible phosphorylation cascade --- p.143 / Chapter 4.2.2. --- Signal transduction potentiated by protein-protein interaction --- p.144 / Chapter 4.3. --- Other signaling molecules --- p.145 / Chapter 4.3.1. --- PRL1-interacting factor G --- p.145 / Chapter 4.3.2. --- Vacuolar-type H+-ATPasen subunit G --- p.146 / Chapter 4.4. --- Regulation of expression of disease resistance candidates --- p.146 / Chapter 4.4.1. --- Transcriptional regulation of disease resistance related genes --- p.146 / Chapter 4.4.1.1. --- G-box binding protein --- p.147 / Chapter 4.4.1.2. --- MYB TF --- p.147 / Chapter 4.4.2. --- Post-transcriptional modification of disease resistance candidates --- p.148 / Chapter 4.4.2.1. --- RNA splicing factor --- p.148 / Chapter 4.4.2.2. --- Glycine rich RNA binding proteins --- p.149 / Chapter 4.4.3. --- Translational regulation of disease resistance related genes --- p.149 / Chapter 4.5. --- Induction of oxidative burst --- p.150 / Chapter 4.6. --- PR proteins --- p.151 / Chapter 4.7. --- Cell maintenance --- p.152 / Chapter 4.7.1. --- Protein folding --- p.152 / Chapter 4.7.2. --- Protein degradation --- p.153 / Chapter 4.7.3. --- ROS scavenging --- p.154 / Chapter 4.7.4. --- Regulation of cell cycle --- p.154 / Chapter 4.8. --- "Confirmation and profiling of disease resistance related candidates commonly regulated in Xa2, Xal2 and Xal4 BLB resistance NILs at different time points" --- p.155 / Chapter 4.8.1. --- Basal resistance related genes --- p.156 / Chapter 4.8.2. --- General disease resistance related genes --- p.161 / Chapter 4.8.3. --- Pathogen responsive genes --- p.164 / Chapter 4.8.4. --- Prediction of novel genes functions --- p.168 / Chapter 4.9. --- Future prospect --- p.169 / Chapter 4.10. --- Conclusion --- p.169 / References --- p.171 / Appendix --- p.187

A doença vassoura-de-bruxa do cacaueiro (Theobroma cacao L.), causada pelo basidiomiceto Moniliophthora perniciosa consiste numa importante enfermidade e apenas o uso de variedades resistentes representa uma solução econômica e ambientalmente viável. Os hormônios vegetais são imprescindíveis na rede de sinalização envolvida na resposta contra uma grande variedade de estresses bióticos e abióticos, sendo bem reconhecido o papel crucial do ácido salicílico (AS), etileno (ET) e os jasmonatos (JA) na interação planta-patógeno. O mecanismo de resistência observado em T. cacao contra o fungo hemibiotrófico M. perniciosa parece não envolver resposta de hipersensibilidade mediada pela sinalização por AS, e caracteriza-se pela menor incidência de sintomas e atenuação do crescimento micelial no material resistente. A resposta regulada por JA e/ou ET é determinada pela contenção e redução da colonização de tecidos infectados pelo patógeno, com atenuação dos sintomas manifestados, e está associada com a indução e produção de inibidores de protease, enzimas líticas da parede de fungos e enzimas do metabolismo secundário e cujo os genes demonstraram indução diferencial em amostras inoculadas com M. perniciosa. Recentemente, foi demonstrada a produção de AS pelo fungo M. perniciosa, o que poderia estar associado a um desarranjo hormonal na planta, auxiliando o pátogeno no processo infectivo. A partir destas evidências este trabalho teve como hipótese que JA e/ou ET estaria regulando a interação T. cacao e M. perniciosa. Sabe-se que a transcrição de genes codificantes das enzimas da via de biossíntese de JA é induzida por aplicação exógena de metil-jasmonato (MJ) e por patógenos, assim para verificar a participação de JA na resposta de defesa de cacau, seqüências de genes que codificam para enzimas da via de biossíntese foram identificadas, classificadas e confirmados sua identidade por seqüenciamento. A indução e expressão quantitativa destes, além dos genes Samsi, Accox, Pal, Jaz e Della, foram avaliadas entre o acesso susceptível à M. perniciosa (\'P7\') e o resistente (\'CAB 214\') de T. cacao, em experimentos de aplicação de indutores (AS, ET e MJ) e inoculação com M. perniciosa. As análises de expressão gênica relativa por RT-qPCR foram conduzidas e a resposta dos genes de biossíntese de JA, quando tratado com MJ no \'P7\' pareceu ser mais intensa e mais específica, enquanto que o acesso \'CAB 214\' apresentou resposta com menor intensidade, porém com resposta mais precoce, demonstrando que o mecanismo de regulação positiva pela aplicação exógena de MJ também ocorre em T. cacao. Em relação à inoculação, os resultados de expressão gênica sugerem uma diferença na resposta transcricional dos genes analisados sob inoculação de M. perniciosa entre o \'P7\' e o \'CAB 214\' onde os transcritos de Aos, Kat, Samsi e Jaz apresentaram elevação significativa somente no \'CAB 214\' em comparação ao \'P7\'. Em acessos resistentes, como \'CAB 214\', o efeito de AS produzido pelo fungo poderia não estar surtindo efeitos antagônicos, como indicado pelo aumento transcricional de Aos, gene codificador da principal enzima envolvida na biossíntese de JA, e embora os demais genes da via estejam sendo reprimidos, muito possivelmente a sinalização da resposta de defesa do acesso resistente \'CAB 214\' seja desencadeada por JA, devido ao papel central de AOS na sua biossíntese, e de maneira sinérgica ET estaria participando do mecanismo de resposta, indicado pela alta indução de Samsi no acesso resistente / Witches broom disease of cacao (Theobroma cacao L.), caused by the basidiomycete Moniliophthora perniciosa is an important disease and the use of resistant varieties is the only economic and environmental long-term solution. Plant hormones are essential in the signaling network involved in the response against a variety of biotic and abiotic stresses. It is well recognized the crucial role of salicylic acid (SA), ethylene (ET) and jasmonate (JA) in plant-pathogen interactions. The mechanism of resistance observed in Theobroma cacao against M. perniciosa does not appear to involve hypersensitivity response mediated by AS signaling, and it is characterized by lower incidence of symptoms and reduction of mycelial growth in resistant material. The response regulated by JA and/or ET is determined by the growth inhibition and a reduction of the colonization of infected tissues by the pathogen, together with an attenuation of symptoms. It is also associated with an induction and production of the protease inhibitors, lytic enzymes and enzymes of secondary metabolism and the genes enconding these enzymes have shown differential expression patterns in samples inoculated with M. perniciosa. It has been recently demonstrated that the production of AS by the fungus M. perniciosa could be associated with a hormonal disorder in the plant, which could therefore help the pathogen in the infective process. Considering this, the hypothesis that JA and/or ET would regulate the interaction of T. cacao with M. perniciosa was formulated in order to be tested by this research work. It is known that the transcription of genes encoding the enzymes of the JA biosynthesis pathway is induced by exogenous application of methyl jasmonate (MJ) and by pathogen, thus, in order to verify the involvement of JA in defense response of cocoa, sequences of genes that encode the enzymes of the JA biosynthesis pathway were isolated, identified, classified and had their identity confirmed by sequencing, and relative quantitative gene expression were evaluated in susceptible \'P7\' and resistant \'CAB 214\' plants of T. cacao. In addition genes Sams, Accox, Pal , Jaz and Della, were evaluated in experiments with application of inducers (AS, ET and MJ) and inoculation with M. perniciosa. Analysis of relative gene expression by RT-qPCR were conducted and \'P7\' seems to have the expression of jasmonate biosynthesis genes in a more intense and more specific manner when treated with MJ, while \'CAB 214\' shows an earlier yet lower response suggesting that the mechanism of positive regulation by the exogenous application of MJ also occurs in T. cacao. For the inoculation, the gene expression results suggest a difference in the transcriptional response from inoculation with M. perniciosa between \'P7\' and \'CAB 214\' in T. cacao. The effect of AS produced by the fungus may not have antagonistic effects in resistant materials such as \'CAB 214\', as indicated by the increase of the transcription of Aos gene that encodes the main enzyme involved in JA biosynthesis, so the defense responses of \'CAB 214\' is possibly triggered by JA signaling, because the central role of AOS in its biosynthesis, and may be part of synergistic ET signaling, indicated by high Samsi expression in resistance material

No description available.

No description available.

Graduation date: 2003

The cereal seed plays a crucial role in society – both in the “food as medicine” paradigm, but also in food security. It is the starch and proteins present in the seed that lend it importance in these dissimilar anthropomorphic activities. This thesis investigation first characterized the post-translational processing of the potential diabetogen, wheat globulin-3. Globulin-3-like peptides were observed primarily in the embryo. These peptides varied significantly in their molecular masses and isoelectric points, as determined by two dimensional electrophoresis and immunoblotting. Five major polypeptide spots were sequenced by mass spectrometry, allowing for the development of a model of the post-translational events contributing to the globulin-3 processing profile. Three separate investigations of starch granules from different cereal species were performed. In the first series of experiments, pathogen-susceptible maize kernels were injected with either conidia of the fungal pathogen Fusarium graminearum or sterile water controls. Proteins in the desiccated fungal remnants on the surface of the kernels as well as in the endosperm and embryo tissues of the control and infected kernels were isolated and these proteomes were sequenced using tandem mass spectrometry. Approximately 250 maize proteins were identified. These proteins were classified into functional categories. There was an increased representation of defense proteins in the both the embryo and endosperm tissues of infected maize samples. The proteome of the fungal remnants was composed of 18 proteins. Several of these proteins were categorized as being involved in the metabolism of plant-sourced molecules, or in stress response. The second series of experiments detail the investigation of commercially prepared rice and maize starches using tandem mass spectrometry. The majority of identified proteins, in both rice and maize samples, were involved in either carbohydrate metabolism or storage. Markers for seed maturity and for starch mobilization were also documented. Finally, the third series of experiments investigated the non-host proteomes present in commercially-prepared starches. Non-host proteins from a variety of species, including Homarus americanus were found in the starch samples. This documentation of H. americanus proteins in these starch samples may have food safety implications with regards to shellfish allergies.