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URI permanente desta comunidadehttps://thoth.dti.ufv.br/handle/123456789/2
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Item Genomic identification, gene expression and enzyme activity of CAZymes from biotrophic fungus Hemileia vastatrix during coffee leaf infection(Universidade Federal de Minas Gerais, 2021-02-25) Pereira, Júlia Santos; Mendes, Tiago Antônio de OliveiraCoffee leaf rust, caused by the fungus Hemileia vastatrix, is a major disease that affects many coffee producers around the world, causing a huge economic loss. H. vastatrix is a biotrophic fungus, hence, its growth and reproduction are totally dependent on the cells of the living host. Because of that, they infect the tissue without causing necrosis. Nevertheless, it is known that some fungi during plant interaction can express genes involved in the formation of infectious structures, as well as synthesize enzymes responsible for the degradation of the host cell wall. However, little is known about the importance of cell wall degrading enzymes (CWDE) for biotrophic phytopathogens. In this work, we performed genomic analysis to identify CAZymes (Carbohydrate Active EnZymes) from H. vastatrix and the analysis of expression and enzyme activity during experimental coffee infection. The first step was the ab initio prediction of H. vastatrix genome, using AUGUSTUS program, which was adjusted with H. vastatrix RNA-Seq for model determination. Functional annotation to identify CAZymes was performed using dbCAN2 meta server. Among the 345 CAZymes found, 162 belong to Glycoside Hydrolases (GH) including 34 endoglucanases, xylanases and polygalacturonases from families GH 5, 9, 10, 12 and 28 - which may act during fungal infection degrading the highest prevalent components of cell wall, represented by cellulose, xylan and pectin, respectively. Physico-chemical parameters of these enzymes were checked, including identification of catalytic residues and standard CAZymes motifs. The enzyme structures were also modelled. Moreover, analysis of expression of enzymes were performed using RNA-seq data from a genotype of C. arabica susceptible to infection, therefore presenting compatible interaction with the pathogen, after 0, 12, 24 and 96 hours after inoculation (hai). Enzyme activity assays were carried out in order to verify if these enzymes were also active during the fungal infection. The enzymes showed typical physico-chemical characteristics and structures of active enzymes including conservation of the catalytic sites. Genes encoding endoglucanases and xylanases were expressed during different times of H. vastatrix infection, being the first more expressed at 12 hai and the other at 24 hai. In agreement with expression data, endoglucanases started to be produced early, and were more active at 24 hai, whereas xylanases had greater activity at 96 hai. The results suggest that biotrophic fungus also use active enzymes on carbohydrates during the infection. In relation to H. vastatrix, these enzymes start being produced at the pre-haustorial phase of the fungal infection, but are more active at the post-haustorial phase, when the haustorium begins to be formed in the host membranes and in the plant cell wall.Item Atividade da celulase sobre o processo germinativo de sementes de cafeeiro (Coffea arabica L.)(Universidade Federal de Lavras, 2002-02-15) Sales, Juliana de Fátima; Alvarenga, Amauri Alves deO presente trabalho foi realizado no Laboratório de Bioquímica e Metabolismo de Plantas e no Laboratório de Análise de Sementes da Universidade Federal de Lavras - MG, tendo como principal objetivo estudar in vitro, as atividades da enzima celulase e sua ação in vivo sobre a germinação de sementes de cafeeiro, visando o aumento do índice de velocidade de germinação e a redução do período de formação das mudas. Por se tratar de um estudo pioneiro relacionado à atividade da enzima celulase sobre a germinação das sementes de cafeeiro, na 1ª fase foi realizado um estudo preliminar com o objetivo de verificar a ação da enzima celulase sobre a decomposição do endocarpo extraído de sementes de cafeeiro. Avaliando a concentração da enzima, a concentração do substrato e o tempo de incubação, foi constatado que a hidrólise do endocarpo in vitro é diretamente proporcional à concentração da enzima celulase utilizada e que a hidrólise do endocarpo cresce linearmente em função do tempo de exposição à enzima. Na 2ª fase, foram realizados 2 experimentos in vivo com imersão das sementes de cafeeiro em diferentes soluções, testando-se várias concentrações da enzima. Foi verificado que sementes sem endocarpo apresentam maiores IVG (Índice de Velocidade de Germinação), IVE (Índice de Velocidade de Emergência), porcentagem de germinação e porcentagem de emergência do que sementes com endocarpo e, também, que sementes com endocarpo imersas por 144 horas em solução de celulase na concentração de 6,4 g.L-¹ apresentam maior IVG e porcentagem de germinação.