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No description available.

Os patógenos de plantas são frequentemente classificados em biotróficos, necrotróficos ou hemibiotróficos de acordo com seu mecanismo de infecção e estilo de vida e, consequentemente, respostas distintas do hospedeiro são ativadas. As respostas da planta a patógenos são reconhecidamente reguladas por hormônios vegetais. Classicamente, considera-se que a sinalização por ácido salicílico (SA) é requerida para resistência a patógenos biotróficos, enquanto que a combinação de ácido jasmônico (JA) e etileno (ET) são requeridos para a resistência a necrotróficos. Com o objetivo de identificar mutantes e/ou transgênicos hormonais que apresentassem resposta diferencial contrastante à infecção pelos patógenos necrotrófico Sclerotinia sclerotiorum e biotrófico Oidium neolycopersici foi realizado um experimento com nove mutantes e transgênicos hormonais de tomateiro juntamente com o controle \'MT\'. Nesse screening foi concluído que o mutante sitiens, com reduzidos níveis de ácido abscísico (ABA), responde diferencialmente, apresentando maior suscetibilidade ao necrotrófico do que \'MT\' e resistência parcial ao fungo biotrófico. Evidências adicionais da participação do ABA nessas interações foram encontradas por meio da detecção no aumento no conteúdo de ABA em folhas de plantas inoculadas com ambos os patógenos, pelo aumento da expressão de GUS no transgênico RD29B::GUS, cujo promotor é responsivo ao ABA , e pela modulação da resistência com a aplicação exógena de 100 μM de ABA ou 2 μM de fluridone em \'MT\', em sitiens e no mutante sp12, com elevados níveis de ABA. Assim, o objetivo deste trabalho foi investigar o papel da via biossintética de ABA na modulação do ciclo ascorbato-glutationa de antioxidação e remoção de H2O2, e do papel do ABA na alteração do perfil transcricional e protéico em folhas de tomateiro em resposta a infecção por S. sclerotiorum ou O. neolycopersici. sitiens apresenta elevados níveis de H2O2 no apoplasto, que parece resultar em morte celular programada (PCD). A presença dessa mutação interrompe a via de biossíntese do ABA, com provável acúmulo de intermediários, ocorrendo o consumo do ascorbato pela enzima violaxantina de-epoxidase (VDE) no cloroplasto. Com isso, esse ascorbato não é transportado para o apoplasto para a remoção de H2O2, o que auxilia no acúmulo de H2O2 e a consequente indução de PCD. A análise da expressão gênica diferencial por RNA-seq permitiu observar o enriquecimento de termos associados a grupos de genes induzidos relacionados a mecanismos de autofagia em \'MT\' inoculado com S. sclerotiorum. A autofagia é um tipo distinto de PCD e pode ser vista como uma estratégia de sobrevivência para evitar a extensiva morte celular, resultando no aumento da resistência a patógenos necrotróficos, como observado em \'MT\'. A análise proteômica permitiu verificar em \'MT\' uma maior abundância relativa de proteínas relacionadas a patogênese quando inoculadas com S. sclerotiorum. As proteínas encontradas em maior abundância relativa estão relacionadas a vias de sinalização do JA e ET, degradação da parede celular de fungos e de metabólitos secundários, conferindo uma maior resistência ao \'MT\'. Na interação com O. neolycopersici, foi verificada uma maior indução dos genes de resistência em sitiens e não foi detectado o enriquecimento de termos ligados a grupos de genes de autofagia em \'MT\'. Por outro lado, a análise de proteínas revelou a participação de uma endoquitinase em sitiens com papel presumível na resistência parcial a este patógeno biotrófico. Em conclusão, a via de biossíntese de ABA pode modificar a capacidade de remoção de H2O2 e ABA pode induzir a formas distintas de morte celular e regular a expressão de genes de resistência. / Plant pathogens are often classified as biotrophic, necrotrophic or hemibiotrophic according to their mechanism of infection and lifestyle and, consequently, distinct host responses are activated in each case. Plant responses to pathogens are known to be regulated by plant hormones. Classically, salicylic acid (SA) signaling is required for resistance to biotrophic pathogens, whereas the combination of jasmonic acid (JA) and ethylene (ET) are required for necrotrophic resistance. In order to identify mutant and/or hormonal transgenic plants that showed differential response to infection by the necrotrophic pathogens Sclerotinia sclerotiorum and biotrophic Oidium neolycopersici, an experiment was carried out with nine mutants and hormonal transgenic tomato plants together with the \'MT\' control. In this screen, the sitiens mutant, with reduced levels of abscisic acid (ABA), presented greater susceptibility to the necrotrophic fungus and partial resistance to the biotrophic fungus in comparison to \'MT\'. Further evidence of the participation of ABA in these interactions was given by the fact that ABA contents increased in leaves of plants inoculated with both pathogens, GUS expression increased in the transgenic RD29B :: GUS whose promoter is responsive to ABA, and resistance was modulated by the exogenous application of 100 μM ABA or 2 μM fluridone in \'MT\', sitiens and the sp12 mutant (high levels of ABA). The objective of this work was to investigate the role of the ABA biosynthetic pathway in the modulation of the ascorbate-glutathione cycle of antioxidation and H2O2 removal, and the role of ABA in altering the transcriptional and protein profile in tomato leaves in response to infection by S. sclerotiorum or O. neolycopersici. sitiens presents high levels of H2O2 in the apoplast, which appears to result in programmed cell death (PCD). The presence of this mutation interrupts the ABA biosynthesis pathway, with a probable accumulation of intermediates, with the use of ascorbate by the enzyme violaxanthin de-epoxidase (VDE) in the chloroplast. Thus, this ascorbate is not transported to the apoplast to remove H2O2, promoting the accumulation of H2O2 and the consequent induction of PCD. In \'MT\' inoculated with S. sclerotiorum, the analysis of differential gene expression by RNA-seq revealed the enrichment of terms associated with groups of induced genes related to mechanisms of autophagy. Autophagy is a distinct type of PCD and is a survival strategy to prevent extensive cell death, resulting in increased resistance to necrotrophic pathogens, as observed in \'MT\'. Proteomic analysis showed that \'MT\' presented a greater relative abundance of proteins related to pathogenesis when inoculated with S. sclerotiorum. These proteins are related to JA and ET signaling pathways, secondary metabolites and cell wall degradation of fungi, conferring greater resistance to \'MT\'. In the interaction with O. neolycopersici, increased induction of resistance genes was observed in sitiens and no enrichment of terms linked to groups of autophagy genes in \'MT\' was detected. On the other hand, protein analysis revealed the participation of an endochitinase in sitiens with a presumed role in the partial resistance to this biotrophic pathogen. In conclusion, the ABA biosynthetic pathway can modify the H2O2 removal capacity and ABA can induce distinct forms of cell death and regulate the expression of resistance genes.

Environmental hazards and health problems due to the application of chemical pesticides in agricultural sectors incite huge public concerns. Therefore, one of the better solutions is through introduction of biological control means to manage the outbreaks of plant diseases. To date, only small numbers of beneficial microorganisms - belonging to the category of hyperparasitic or mycoparasitic fungi have proven to keep plants or protect crops from plant pathogen infection. The objective of this study was to characterize a group of <i>Fusarium</i>-associated melanosporaceous biotrophic mycoparasitic fungal isolates, which were identified and pre-selected by Dr. Vladimir Vujanovic and deposited in the Saskatchewan Microbial Collection and Database (SMCD). Particular objectives were to examine spore germination of a biotrophic mycoparasite, to test effects of this fungus on seed germinations, to study interactions between the mycoparasite and Fusarium hosts, and to investigate relationships between the mycoparasite-the <i>Fusarium</i> host-wheat root under controlled conditions in the university Phytotron facilities.<p> Information related to this group of fungi is relatively limited. In order to characterize potential biotrophic mycoparasitic fungal isolate(s), molecular and microscopy methods were performed to accomplish taxonomical, phylogenetical and morphological studies. Since, spore germination is a very crucial stage in fungal life cycle and growth, ascospores (sexual spores) of the biotrophic mycoparasite were isolated from a fungal colony. These spores were inoculated on media supplemented with different <i>Fusarium</i>-filtrates or suspended in different <i>Fusarium</i>-filtrates to examine spore germination rates and growth patterns. Together with other mycoparasitic fungi, this biotrophic mycoparasite was inoculated on spring wheat seeds, to test effects of these fungal inoculants on seedlings growth using <i>in vitro</i> assays. Dual-culture, slide culture, and microscopy approaches were carried out to elucidate intimate and special relationship between the biotrophic mycoparasite and <i>Fusarium</i>-hosts. In order to study tritrophic interactions (biotrophic mycoparasite-<i>Fusarium</i> host-wheat root), spring wheat was grown in the phytotron with different treatments of fungal inoculations. Wheat roots were then subjected to genus-specific quantitative real-time PCR analyses.<p> One melanosporaceous biotrophic mycoparasitic strain was identified as a new species in the genus <i>Sphaerodes</i>. This biotrophic mycoparasite was isolated from <i>Fusarium</i>-infected fields in Saskatchewan and Quebec, and named <i>Sphaerodes mycoparasitica</i>. Germination of <i>S. mycoparasitica</i> sexual spores was improved when treated with filtrates or extracellular extracts from the <i>Fusarium</i>-host as compared to <i>Fusarium</i>-non-host filtrates. No pathogenic effects on wheat seeds were observed when inoculated with <i>S. mycoparasitica</i>. Furthermore, seedlings growth was enhanced with this biotrophic mycoparasite compared to other mycoparasitic fungi. Later, this biotrophic mycoparasitic strain was found to establish biotrophic fusion and haustorial contact relations with <i>F. avenaceum, F. oxysporum</i>, and two F. graminearum chemotypes. Since, 3-Acetyldeoxynivalenol-producing <i>F. graminearum</i> is one of the most highly toxigenic and aggressive wheat pathogens in Saskatchewan and North America, therefore, this pathogen strain was chosen for tritrophic interaction study. Under controlled conditions in the phytotron, <i>S. mycoparasitica</i> improved seedlings growth when these were challenged with <i>F. graminearum</i> as compared to seedlings only inoculated with the <i>Fusarium</i> pathogen. In conclusion, S. mycoparasitica could be a potential candidate for biological control of <i>Fusarium</i> diseases in wheat.

Environmental hazards and health problems due to the application of chemical pesticides in agricultural sectors incite huge public concerns. Therefore, one of the better solutions is through introduction of biological control means to manage the outbreaks of plant diseases. To date, only small numbers of beneficial microorganisms - belonging to the category of hyperparasitic or mycoparasitic fungi have proven to keep plants or protect crops from plant pathogen infection. The objective of this study was to characterize a group of <i>Fusarium</i>-associated melanosporaceous biotrophic mycoparasitic fungal isolates, which were identified and pre-selected by Dr. Vladimir Vujanovic and deposited in the Saskatchewan Microbial Collection and Database (SMCD). Particular objectives were to examine spore germination of a biotrophic mycoparasite, to test effects of this fungus on seed germinations, to study interactions between the mycoparasite and Fusarium hosts, and to investigate relationships between the mycoparasite-the <i>Fusarium</i> host-wheat root under controlled conditions in the university Phytotron facilities.<p> Information related to this group of fungi is relatively limited. In order to characterize potential biotrophic mycoparasitic fungal isolate(s), molecular and microscopy methods were performed to accomplish taxonomical, phylogenetical and morphological studies. Since, spore germination is a very crucial stage in fungal life cycle and growth, ascospores (sexual spores) of the biotrophic mycoparasite were isolated from a fungal colony. These spores were inoculated on media supplemented with different <i>Fusarium</i>-filtrates or suspended in different <i>Fusarium</i>-filtrates to examine spore germination rates and growth patterns. Together with other mycoparasitic fungi, this biotrophic mycoparasite was inoculated on spring wheat seeds, to test effects of these fungal inoculants on seedlings growth using <i>in vitro</i> assays. Dual-culture, slide culture, and microscopy approaches were carried out to elucidate intimate and special relationship between the biotrophic mycoparasite and <i>Fusarium</i>-hosts. In order to study tritrophic interactions (biotrophic mycoparasite-<i>Fusarium</i> host-wheat root), spring wheat was grown in the phytotron with different treatments of fungal inoculations. Wheat roots were then subjected to genus-specific quantitative real-time PCR analyses.<p> One melanosporaceous biotrophic mycoparasitic strain was identified as a new species in the genus <i>Sphaerodes</i>. This biotrophic mycoparasite was isolated from <i>Fusarium</i>-infected fields in Saskatchewan and Quebec, and named <i>Sphaerodes mycoparasitica</i>. Germination of <i>S. mycoparasitica</i> sexual spores was improved when treated with filtrates or extracellular extracts from the <i>Fusarium</i>-host as compared to <i>Fusarium</i>-non-host filtrates. No pathogenic effects on wheat seeds were observed when inoculated with <i>S. mycoparasitica</i>. Furthermore, seedlings growth was enhanced with this biotrophic mycoparasite compared to other mycoparasitic fungi. Later, this biotrophic mycoparasitic strain was found to establish biotrophic fusion and haustorial contact relations with <i>F. avenaceum, F. oxysporum</i>, and two F. graminearum chemotypes. Since, 3-Acetyldeoxynivalenol-producing <i>F. graminearum</i> is one of the most highly toxigenic and aggressive wheat pathogens in Saskatchewan and North America, therefore, this pathogen strain was chosen for tritrophic interaction study. Under controlled conditions in the phytotron, <i>S. mycoparasitica</i> improved seedlings growth when these were challenged with <i>F. graminearum</i> as compared to seedlings only inoculated with the <i>Fusarium</i> pathogen. In conclusion, S. mycoparasitica could be a potential candidate for biological control of <i>Fusarium</i> diseases in wheat.

Os patógenos de plantas são frequentemente classificados em biotróficos, necrotróficos ou hemibiotróficos de acordo com seu mecanismo de infecção e estilo de vida e, consequentemente, respostas distintas do hospedeiro são ativadas. As respostas da planta a patógenos são reconhecidamente reguladas por hormônios vegetais. Classicamente, considera-se que a sinalização por ácido salicílico (SA) é requerida para resistência a patógenos biotróficos, enquanto que a combinação de ácido jasmônico (JA) e etileno (ET) são requeridos para a resistência a necrotróficos. Com o objetivo de identificar mutantes e/ou transgênicos hormonais que apresentassem resposta diferencial contrastante à infecção pelos patógenos necrotrófico Sclerotinia sclerotiorum e biotrófico Oidium neolycopersici foi realizado um experimento com nove mutantes e transgênicos hormonais de tomateiro juntamente com o controle \'MT\'. Nesse screening foi concluído que o mutante sitiens, com reduzidos níveis de ácido abscísico (ABA), responde diferencialmente, apresentando maior suscetibilidade ao necrotrófico do que \'MT\' e resistência parcial ao fungo biotrófico. Evidências adicionais da participação do ABA nessas interações foram encontradas por meio da detecção no aumento no conteúdo de ABA em folhas de plantas inoculadas com ambos os patógenos, pelo aumento da expressão de GUS no transgênico RD29B::GUS, cujo promotor é responsivo ao ABA , e pela modulação da resistência com a aplicação exógena de 100 &mu;M de ABA ou 2 &mu;M de fluridone em \'MT\', em sitiens e no mutante sp12, com elevados níveis de ABA. Assim, o objetivo deste trabalho foi investigar o papel da via biossintética de ABA na modulação do ciclo ascorbato-glutationa de antioxidação e remoção de H2O2, e do papel do ABA na alteração do perfil transcricional e protéico em folhas de tomateiro em resposta a infecção por S. sclerotiorum ou O. neolycopersici. sitiens apresenta elevados níveis de H2O2 no apoplasto, que parece resultar em morte celular programada (PCD). A presença dessa mutação interrompe a via de biossíntese do ABA, com provável acúmulo de intermediários, ocorrendo o consumo do ascorbato pela enzima violaxantina de-epoxidase (VDE) no cloroplasto. Com isso, esse ascorbato não é transportado para o apoplasto para a remoção de H2O2, o que auxilia no acúmulo de H2O2 e a consequente indução de PCD. A análise da expressão gênica diferencial por RNA-seq permitiu observar o enriquecimento de termos associados a grupos de genes induzidos relacionados a mecanismos de autofagia em \'MT\' inoculado com S. sclerotiorum. A autofagia é um tipo distinto de PCD e pode ser vista como uma estratégia de sobrevivência para evitar a extensiva morte celular, resultando no aumento da resistência a patógenos necrotróficos, como observado em \'MT\'. A análise proteômica permitiu verificar em \'MT\' uma maior abundância relativa de proteínas relacionadas a patogênese quando inoculadas com S. sclerotiorum. As proteínas encontradas em maior abundância relativa estão relacionadas a vias de sinalização do JA e ET, degradação da parede celular de fungos e de metabólitos secundários, conferindo uma maior resistência ao \'MT\'. Na interação com O. neolycopersici, foi verificada uma maior indução dos genes de resistência em sitiens e não foi detectado o enriquecimento de termos ligados a grupos de genes de autofagia em \'MT\'. Por outro lado, a análise de proteínas revelou a participação de uma endoquitinase em sitiens com papel presumível na resistência parcial a este patógeno biotrófico. Em conclusão, a via de biossíntese de ABA pode modificar a capacidade de remoção de H2O2 e ABA pode induzir a formas distintas de morte celular e regular a expressão de genes de resistência. / Plant pathogens are often classified as biotrophic, necrotrophic or hemibiotrophic according to their mechanism of infection and lifestyle and, consequently, distinct host responses are activated in each case. Plant responses to pathogens are known to be regulated by plant hormones. Classically, salicylic acid (SA) signaling is required for resistance to biotrophic pathogens, whereas the combination of jasmonic acid (JA) and ethylene (ET) are required for necrotrophic resistance. In order to identify mutant and/or hormonal transgenic plants that showed differential response to infection by the necrotrophic pathogens Sclerotinia sclerotiorum and biotrophic Oidium neolycopersici, an experiment was carried out with nine mutants and hormonal transgenic tomato plants together with the \'MT\' control. In this screen, the sitiens mutant, with reduced levels of abscisic acid (ABA), presented greater susceptibility to the necrotrophic fungus and partial resistance to the biotrophic fungus in comparison to \'MT\'. Further evidence of the participation of ABA in these interactions was given by the fact that ABA contents increased in leaves of plants inoculated with both pathogens, GUS expression increased in the transgenic RD29B :: GUS whose promoter is responsive to ABA, and resistance was modulated by the exogenous application of 100 &mu;M ABA or 2 &mu;M fluridone in \'MT\', sitiens and the sp12 mutant (high levels of ABA). The objective of this work was to investigate the role of the ABA biosynthetic pathway in the modulation of the ascorbate-glutathione cycle of antioxidation and H2O2 removal, and the role of ABA in altering the transcriptional and protein profile in tomato leaves in response to infection by S. sclerotiorum or O. neolycopersici. sitiens presents high levels of H2O2 in the apoplast, which appears to result in programmed cell death (PCD). The presence of this mutation interrupts the ABA biosynthesis pathway, with a probable accumulation of intermediates, with the use of ascorbate by the enzyme violaxanthin de-epoxidase (VDE) in the chloroplast. Thus, this ascorbate is not transported to the apoplast to remove H2O2, promoting the accumulation of H2O2 and the consequent induction of PCD. In \'MT\' inoculated with S. sclerotiorum, the analysis of differential gene expression by RNA-seq revealed the enrichment of terms associated with groups of induced genes related to mechanisms of autophagy. Autophagy is a distinct type of PCD and is a survival strategy to prevent extensive cell death, resulting in increased resistance to necrotrophic pathogens, as observed in \'MT\'. Proteomic analysis showed that \'MT\' presented a greater relative abundance of proteins related to pathogenesis when inoculated with S. sclerotiorum. These proteins are related to JA and ET signaling pathways, secondary metabolites and cell wall degradation of fungi, conferring greater resistance to \'MT\'. In the interaction with O. neolycopersici, increased induction of resistance genes was observed in sitiens and no enrichment of terms linked to groups of autophagy genes in \'MT\' was detected. On the other hand, protein analysis revealed the participation of an endochitinase in sitiens with a presumed role in the partial resistance to this biotrophic pathogen. In conclusion, the ABA biosynthetic pathway can modify the H2O2 removal capacity and ABA can induce distinct forms of cell death and regulate the expression of resistance genes.

Doctor of Philosophy / Department of Plant Pathology / Barbara S. Valent / The hemibiotrophic rice blast fungus Magnaporthe oryzae undergoes complex morphological development throughout its infection cycle. From 8-20 hours after a fungal spore lands on a leaf surface, the fungus differentiates a complex appressorium that punctures the host cuticle. By ~24 hours post inoculation (hpi), the fungus grows inside an epidermal cell as a primary hypha, and by 36 hpi the fungus has differentiated specialized biotrophic invasive hyphae (IH) that are filling the first-invaded cell and moving into neighbor cells. Throughout its life cycle, IH invade living rice cells although invaded cells appear dead when the fungus moves into the next cell. Biotrophic invasion must be mediated by fungal effectors, proteins that pathogens secrete inside live host cells to control them. However, little is known about blast effectors, and the low fungal biomass in early infection stages complicates identification of effector genes, as well as identification of rice genes controlled by effectors. The characterized AVR-Pita effector gene is specifically expressed in planta, but it was not clear how its gene expression pattern changed in different infection stages. We found that AVR-Pita is first expressed around the time of penetration. AVR-Pita is highly expressed in IH developing in asymptomatic tissue from 36 hpi to as late as 7 days post inoculation when lesions are maturing. Using inoculated rice sheaths, we successfully enriched for infected tissue RNA that contained ~20% IH RNA at 36 hpi. We compared IH gene expression to expression in mycelium from pure culture using a whole-genome M. oryzae oligoarray, and we compared infected rice gene expression to expression in mock-inoculated tissue using a rice oligoarray. Rice genes that were induced >50-fold during infection were enriched for genes involved in transferring information from sensors to cellular responses. Fungal genes that were induced >50-fold in IH included known effectors and many IH-specific genes encoding hypothetical secreted proteins that are candidate effectors. Gene knock-out analyses of three putative effector genes failed to show major effects on pathogenicity. Details of the blast interaction transcriptome will provide insights on the mechanisms of biotrophic plant disease.

Austropuccinia psidii, the causal agent of myrtle rust, is a biotrophic pathogen, and therefore its growth and development depend on the host tissues. The uredospores of A. psidii infect Eucalyptus by engaging in close contact with the host surface and interacting with the leaf cuticle that provides important chemical and physical signals to trigger the infection process. Due to the inherent characteristics of the Eucalyptus cuticle, it was hypothesized that the preformed mechanism, comprised mostly by cuticular waxes, plays a crucial role in Eucalyptus resistance against A. psidii and its ability to modulate the expression of genes associated to the pathogenicity of A. psidii during the early stage of infection. In chapter 2, the cuticular waxes of Eucalyptus spp. were analyzed to determine their composition or structure and then correlated to susceptibility/resistance to Austropuccinia psidii. Twenty-one Eucalyptus spp. in the field were classified as resistant or susceptible. From these, the resistance/susceptibility level of six Eucalyptus spp. was evaluated in controlled conditions using qPCR, revealing that the pathogen can germinate on the eucalyptus surface of some species without multiplying in the host. CG-TOF-MS analysis detected 26 compounds in the Eucalyptus spp. cuticle and led to the discovery of the role of hexadecanoic acid in the susceptibility of E. grandis and E. phaeotricha to A. psidii. The scanning electron microscopy check revealed differences in A. psidii germination during host infection. It was found a correlation between epicuticular morphology and the resistance to A. psidii. In chapter 3, we investigated gene expression of A. psidii through bioassays in vitro containing cuticular waxes from E. grandis (E. g), E. urograndis (E. ug) and E. urophylla (E. u). Mineral oil (MO) treatment was used to all comparative analysis (negative control). The presence of cuticular waxes from E. g induced the expression of genes encoding proteins related to growth and colonization of A. psidii such as binding proteins (peptidylprolyl isomerase and ribosomal) and cell wall degrading proteins (beta-xylanase). However, other pathogenic proteins were repressed in presence of cuticular wax of E. g, for instance, triosephosphate isomerase, family 18 glycoside hydrolase, mitochondrial ATP carrier, and glutamine-dependent NAD synthetase. The E. ug x MO analysis resulted in DEGs associated with proteins related to membrane transporters and receptors, DNA repair and glycine dehydrogenase. As to the cuticular wax of E. u, it up-regulated the expression of genes encoding proteins associated with pheromone, cutinases, and prefoldin. Thus, for the first time, it was demonstrated a considerable interspecific variation in Eucalyptus species on the susceptibility to A. psidii and its correlation with cuticular waxes chemical compounds that seem to play a synergistic role as a preformed defense mechanism. We also demonstrated that Eucalyptus spp. cuticular waxes may modulate the A. psidii gene expression, suggesting the importance of early plant-pathogen molecular interaction to the development of myrtle rust. / Austropuccinia psidii é o agente causal da ferrugem das mirtáceas com crescimento biotrófico, ou seja, o patógeno depende dos tecidos do hospedeiro para crescer e se desenvolver. Os uredósporos de A. psidii infectam Eucalyptus por meio do contato inicial com a superfície do hospedeiro e também pela interação com a cutícula da folha que por sua vez fornece importantes sinais químicos e físicos capaz de desencadear o processo de infecção. Devido às características inerentes à cutícula de Eucalyptus, consideramos as hipóteses de que o mecanismo pré-formado, composto principalmente pelas ceras cuticulares, desempenha um papel crucial na resistência de Eucalyptus spp. contra A. psidii, e, também, é capaz de modular a expressão fúngica de genes associados a patogenicidade durante o estágio inicial de infecção de A. psidii. No capítulo 2, as ceras cuticulares de Eucalyptus spp. foram analisadas para determinar a composição/estrutura e sua correlação com suscetibilidade/resistência de A. psidii. Vinte e uma espécies de Eucalyptus foram classificadas em campo como resistentes ou suscetíveis. A análise de qPCR de seis Eucalyptus spp. revelou que o patógeno pode germinar na superfície de algumas espécies de eucaliptos sem se multiplicar no tecido hospedeiro. Foram identificados 26 compostos presentes na cutícula de Eucalyptus spp. e descobrimos o papel do ácido hexadecanóico na suscetibilidade de E. grandis e E. phaeotricha à ferrugem. Por meio da microscopia eletrônica de varredura encontramos uma correlação entre a morfologia epicuticular e a resistência contra A. psidii. No capítulo 3 para compreender a expressão gênica de A. psidii realizamos bioensaios (in vitro) contendo as ceras cuticulares de E. grandis (E. g), E. urograndis (E. ug) e E. urophylla (E. u). O tratamento com óleo mineral (MO) foi utilizado em todas as análises comparativas como controle negativo. A presença de ceras cuticulares de E. g induziu a expressão de genes que codificam proteínas relacionadas ao crescimento e colonização de A. psidii, como proteínas de ligação (peptidylprolyl isomerase e ribosomal) e proteínas de degradação da parede celular (beta xilanase). No entanto, outras proteínas patogênicas foram reprimidas na presença da cera cuticular de E. g, por exemplo, triosephosphate isomerase, family 18 glycoside hydrolase, mitochondrial ATP carrier e glutamine-dependent NAD synthetase. A análise de E. ug x MO resultou na ativação de proteínas associadas a transportadores e receptores de membrana, reparo de DNA e glycine dehydrogenase. Já a cera cuticular de E. u induziu a expressão de genes que codificam proteínas associadas a feromônios, cutinases e prefoldin. Pela primeira vez, está sendo apresentado a considerável variação interespecífica em espécies de Eucalyptus quanto à suscetibilidade a ferrugem, e, sua correlação com os compostos químicos de ceras cuticulares, os quais parecem ser um importante mecanismo de defesa pré-formado. Também foi revelado que as ceras cuticulares de Eucalyptus spp. são capazes de modular a expressão gênica de A. psidii, evidenciando o papel da interação molecular planta-patógeno precoce no desenvolvimento da ferrugem das mirtáceas.

A cultura do eucalipto apresenta grande importância no setor florestal no mundo. No Brasil, 70% da área florestal plantada é destinada ao eucalipto. Entretanto, a ferrugem das mirtáceas, também conhecida como ferrugem do eucalipto, causada pelo fungo Puccinia psidii Winter, afeta o enorme potencial produtivo das plantações de eucalipto. A biologia, mecanismos de patogenicidade e genética desse patógeno são pouco conhecidos, apesar de sua importância para o setor florestal. Os elementos transponíveis (TEs) são sequências de DNA com a capacidade de migrar e influenciar a organização, integridade e evolução do genoma hospedeiro. O presente trabalho teve como principal objetivo estudar os TEs presentes no genoma de P. psidii, combinando ferramentas in silico e moleculares. A classificação dos elementos transponíveis no genoma de P. psidii MF-1 foi realizada utilizando contigs previamente minerados e remontados, bem como sem seleção prévia dos contigs, por meio do programa RepeatMasker. Ambas estratégias apontaram o predomínio de elementos da Classe I - LTR Retrotransposons no genoma de P. psidii MF-1. O resultado condiz com a composição de TEs em fungos fitopatogênicos descrita na literatura. Algumas análises in silico, como verificação de integridade e anotação manual de sequências proteicas foram também realizadas para alguns contigs classificados como TEs. Assim, foi possível observar a presença de sequências conservadas pertencentes à região pol em LTR Retrotransposons. Além disso, as análises permitiram inferir sobre a existência de TEs híbridos no genoma parcialmente sequenciado de P. psidii MF-1. Paralelamente foi também realizada uma análise comparativa entre os TEs presentes nos genomas de P. graminis, P. striiformis, P. triticina e P. psidii. Observou-se que P. graminis, P. striiformis e P. triticina apresentam maior frequência de elementos da Classe II, do tipo DNA Transposons ao contrário de P. psidii, com maior frequência de elementos da Classe I. Interessantemente, a quantidade de elementos desconhecidos foi similarmente alta para todos os quatro genomas avaliados. Este tipo de análise é muito importante, pois evidencia a grande quantidade de famílias de TEs novas a serem descobertas. Elas podem estar potencialmente relacionadas ao silenciamento de genes importantes à virulência destes patógenos. A utilização de TEs no estudo de diversidade genética entre populações é bastante comum. A técnica molecular IRAP foi utilizada para acessar a diversidade entre populações de P. psidii originárias de três híbridos de Eucalyptus spp., goiabeira, jambeiro e jabuticabeira. No entanto, esta técnica não se mostrou eficiente para detectar polimorfismos existentes entre estas populações. A anotação de TEs foi difícil devido à observação de sequências de elementos sobrepostas, o que podem representar híbridos de TEs, entretanto, visando a confirmação desta hipótese por meio da PCR, alguns contigs serão sequenciados e mais estudos devem ser realizados para a continuação desta confirmação. Os resultados apresentados neste trabalho são inéditos e representam uma etapa crucial no entendimento de TEs em fungos do gênero Puccinia, em especial do patógeno P. psidii para o desenvolvimento de melhores mecanismos de controle de ferrugem. / The culture of eucalyptus has great importance worldwide in forestry sector. In Brazil, 70% of cultivated forest area is intended for Eucalyptus. However, the eucalyptus potential productive has been affected by rust disease, caused by the fungus Puccinia psidii Winter. Despite its importance to brazilian and world forest sector, the knowledge of biology, genetic and pathogenic mechanisms of this pathogen is scarce. Transposable elements (TEs) are mobile DNA fragments that influence the organization and development of the host genome. These elements have the ability to move within host genome, and their insertion can cause a wide spectrum of mutations in their hosts. This study aims to decipher the TEs in P. psidii genome by combining in silico and molecular tools. P. psidii MF-1 TEs classification was performed automatically, through RepeatMasker software, being observed a predominance of Class I - LTR Retrotransposons in P. psidii MF-1 genome. This result is consistent with the TEs composition described in phytopathogenic fungi. Some in silico analysis, as integrity and manual annotation of conserved protein sequences from TEs were carried out with P. psidii MF-1 contigs classified as transposable elements. The presence of conserved sequences belonging to pol region in LTR Retrotransposons was observed. Furthermore, these analysis allowed the inference of hybrid TEs in P. psidii MF-1. At the same time, a comparative analysis of TEs present in other Puccinia genomes and P. psidii MF-1 was also performed. The P. graminis, P. striiformis and P. triticina genomes have higher frequency of Class II - DNA Transposons unlike the results found for P. psidii. Interestingly, the number of unknown elements was similarly high for all genomes. This type of analysis is very importante because it shows a great number of potential new TEs families to be discovered. They may be potentially related to the virulence gene silencing of these pathogens. Using TEs for study the fungal genetic diversity is quite common. The IRAP technique was used to access the diversity among P. psidii populations originated from three Eucalyptus spp. hybrids, guava, syzigium and jabuticaba. However, this technique was not efficient to detect existing polymorphisms between these populations. TEs annotation was labored due to the existence of overlapping elements, which may represent hybrids TEs. PCR tool was used to confirm some sequences annotated as hybrids and more studies are needed to confirm this hyphotesis. The results presented in this study are novel and is a crucial step in understanding the genetic of P. psidii pathogen for further improvements of rust control mechanisms.

Made available in DSpace on 2016-02-26T12:59:37Z (GMT). No. of bitstreams: 1 GilbertoFloresSouza.pdf: 4776672 bytes, checksum: b8aabb595b89b1322dab91e3d6af328d (MD5) Previous issue date: 2007-03-29 / Soybean yield has been the main culture in Brazil for its cultivation area and grain yield, which puts Brazil in the second place in world greatest yield and in the first place in country exporter. Although, the potential of productive efficiency of that culture of 4,000 kg hectare, it is hart to reach. Among main factors, diseases predominated, with great importance for Asian rust that is caused by Phakopsora pachyrhizi Syd. & Syd. That disease is considered as one of the most destroyer disease due to its aggressively and rapid dissemination. As it is a biotrophic pathogen, multiplication and surviving of that fungus only occur in live tissues of plants, this way, tiguera soybean and alternative hosts can allow the surviving of the pathogen, which produces the initial inoculum for subsequent epidemicals. The present work aimed to evaluating the susceptible of nine species of leguminous as alternative hosts of P. pachyrhizi Syd. & P. Syd, the fungus that causes Asian rust. This work was carried out in two periods, one in B.O.D. chamber, temperature of 25ºC and R.H. 93%, and other at laboratory at 20(+ or -2)°C, both two periods at Unit II of the Federal University of Great Dourados, Agrarian Science Institute, from March, 2006, to February, 2007. The following species were evaluated as green manure: common vetch (Vicia sativa L), green bean (Vigna unguiculata L.) Walp, fedegoso (Senna occidentalis) L., sunnhep (Crotalaria juncea L.), common bean (Phaseolus vulgaris L.), velvetbean (Mucuna aterrima L.) lablab bean (Dolichos lablab L.), jack bean (Canavalia ensiformes D.C.) and pea (Pisum sativum L). Every species that were inoculated with uredospore of P. pachyryhizi Syd. & P. Syd developed the symptoms; although, some species showed varied symptoms regarding to the number of harms and the presence of uredospore. Soybean, common bean, lablab bean and velvetbean showed fungal structures (uredospore) in both two conditions that the experiment was carried out, which shows that they have potential to hosting P. pachyrhizi Syd. & Syd. Jack bean, sunnhep, green bean and common vetch were considered susceptible plants, although those species showed be more exigent for sporulation of fungus / A produção de soja (Glycine max (L) Merrill) se tornou a principal cultura brasileira em área cultivada e produção de grãos, colocando o Brasil na posição de segundo maior produtor mundial e primeiro país exportador. Não obstante, o potencial de rendimento dessa cultura de 4.000 kg por hectare, é dificilmente alcançado. Dentre os principais fatores destacam-se as doenças; com elevado destaque para a ferrugem asiática causada por Phakopsora pachyrhizi Syd. & Syd. Essa doença é considerada como uma das mais destrutivas, devido sua agressividade e rápida disseminação. Por se tratar de um patógeno biotrófico, a multiplicação e sobrevivência do fungo só ocorrem em tecidos vivos de plantas, assim sendo, a soja tigüera e os hospedeiros alternativos podem permitir a sobrevivência do patógeno, produzindo o inóculo inicial para as epidemias subseqüentes. O presente trabalho objetivou avaliar a suscetibilidade de nove espécies de leguminosas como hospedeiras alternativas do P. pachyrhizi Syd. & P. Syd, o fungo causador da ferrugem asiática. Este trabalho foi desenvolvido em duas etapas, uma em B. O. D., temperatura de 25° C e U. R. 93% , e a outra no laboratório a 20 (+ ou -2) ° C, ambas na Unidade II da Universidade Federal da Grande Dourados, dependências da Faculdade de Ciências Agrárias, no período de março 2006 a fevereiro de 2007. Foram avaliadas as seguintes espécies utilizadas como adubo verde: ervilhaca (Vicia sativa L), feijão miúdo (Vigna unguiculata L). Walp, fedegoso (Senna occidentalis) L, crotalária (Crotalaria juncea L.), feijão comum (Phaseolus vulgaris L.), mucuna preta (Mucuna aterrima L.), labe-labe (Dolichos lablab L.), feijão de porco (Canavalia ensiformes D.C.) e ervilha (Pisum sativum L). Todas as espécies inoculadas com uredosporos do P. pachyrhizi Syd. & P. Syd, desenvolveram sintomas; no entanto, algumas espécies apresentaram sintomatologia variada quanto ao número de lesões e a presença de uredosporos. A soja, feijão comum, labe-labe e mucuna preta apresentaram estruturas fúngicas (uredosporos), nas duas condições em que foi desenvolvido este experimento, demonstrando terem potencial para hospedar o P. pachyrhizi Syd. & Syd. O feijão de porco, crotalária, feijão miúdo e ervilha foram considerados plantas suscetíveis, porém, estas espécies demonstraram serem mais exigentes para a esporulação do fungo