Browsing by Author "Costa, Rodrigo Arthur Fonseca"
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- Cloning and expression analysis of an endo‑1,3‑β‑d‑glucosidase from Phytophthora cinnamomiPublication . Costa, Rodrigo Arthur Fonseca; Choupina, Altino; Dominguez, ÁngelPhytophthora is considered one of the most destructive genus for many agricultural plant species worldwide, with a strong environmental and economic impact. Phytophthora cinnamomi is a highly aggressive Phytophthora species associated with the forest decline and responsible for the ink disease in chestnut trees (Castanea sativa Miller), a culture which is extremely important in Europe. This pathogenicity occurs due to the action of several enzymes like the hydrolysis of 1,3-β-glucans at specific sites by the enzyme endo-1,3-β-d-glucosidase. The aim of this work to analyze the heterologous expression in two microorganisms, Escherichia coli and Pichia pastoris, of an endo-1,3-β-d-glucosidase encoded by the gene ENDO1 (AM259651) from P. cinnamomi. Different plasmids were used to clone the gene on each organism and the real-time quantitative polymerase chain reaction was used to determine its level of expression. Homologous expression was also analyzed during growth in different carbon sources (glucose, cellulose, and sawdust) and time-course experiments were used for endo-1,3-β-d-glucosidase production. The highest expression of the endo-1,3-β-d-glucosidase gene occurred in glucose after 8 h of induction. In vivo infection of C. sativa by P. cinnamomi revealed an increase in endo-1,3-β-d-glucosidase expression after 12 h. At 24 h its expression decreased and at 48 h there was again a slight increase in expression, and more experiments in order to further explain this fact are underway.
- Cloning, characterization, in vitro and in planta expression of a necrosis‑inducing Phytophthora protein 1 gene npp1 from Phytophthora cinnamomiPublication . Martins, Ivone; Meirinho, Sofia G.; Costa, Rodrigo Arthur Fonseca; Cravador, Alfredo; Choupina, AltinoThe soil-borne oomycete Phytophthora cinnamomi is a highly destructive Phytophthora species associated with the decline of forest. This pathogen secretes a novel class of necrosis-inducing proteins known as Nep1-like proteins (NLPs). In this work, we report the sequencing and molecular characterization of one of these proteins, more specifically the necrosis-inducing Phytophthora protein 1 (NPP1). The ORF of the npp1 gene (EMBL database AM403130) has 768 bp encoding a putative peptide of 256 amino acids with a molecular weight of approximately 25 kD. In order to understand its function, in vitro gene expression was studied during growth in different carbon sources (glucose, cellulose, and sawdust), and at different times of infection, in vivo by RT-qPCR. The highest expression of the npp1 gene occurred in glucose medium followed by sawdust. In vivo infection of Castanea sativa roots with P. cinnamomi revealed a decrease in npp1 expression from 12 to 24 h; at 36 h its expression increased suggesting the existence of a complex mechanism of defense/attack interaction between the pathogen and the host. Expression of recombinant npp1 gene was achieved in Pichia pastoris and assessed by SDS-PAGE analysis of the protein secreted into the culture supernatant, revealing the presence of the NPP1 protein.
- Determination of the subcellular localisation of GIP and NPP1 proteins in Phytophtora cinnamomi tagged with green fluorescent proteinPublication . Chahed, Abdessalem; Chatti, Noureddine; Costa, Rodrigo Arthur Fonseca; Sousa, Maria João; Choupina, AltinoP. cinnamomi is considered one of the world's worst invasive pathogens with a serious threat to a wide range of plant species throughout the world. These species cause several billions of dollars of damage on crop, ornamental, and native plants and so far no efficient solutions have been found to control the disease. A notable characteristic of the interaction between plants and their microbial pathogens is the secretion by both partners of proteins that are associated with attack, defense, and counterdefense thus it’s fundamental to improve our knowledge about these proteins with the intention of finding a solution to the disease affecting the Chestnut.
- Diversidade da microbiota de fungos da própolis in naturaPublication . Costa, Rodrigo Arthur Fonseca; Bispo, Aline Simões da Rocha; Choupina, Altino; Evangelista-Barreto, Norma Suely; Carvalho, Carlos Alfredo Lopes de; Estevinho, Leticia M.; Sodré, Geni da SilvaO presente estudo teve por objetivo realizar a identificação dos fungos encontrados na própolis produzido por Apis mellifera L. da Baía do Iguape, Brasil. Para tanto, foram utilizadas técnicas morfológicas, bioquímicas e moleculares, sendo averiguado o perfil de restrição gerado por espaçador interno transcrito (ITS1 e ITS4). O tamanho dos produtos de PCR foi analisado quanto ao perfil de restrição obtidos com endonuclease (HhaI, HaeIII e HinfI) por espécie. Foram identificadas dezesseis espécies de fungos filamentosos: Flavodon flavus, Aspergillus nomius, Aspergillus versicolor, Cladosporium sp., Coniothyrium sidae, Didymella sp., Paecilomyces variotii, Cladosporium cladosporioides, Penicillium citrinum, Fusarium incarnatum, Penicillium chermesinum, Phoma sp., Stagonosporopsis valerianellae, Phoma medicaginis, Paraphoma fimeti e Stagonosporopsis cucurbitacearum; e seis espécies de leveduras: Candida tropicalis, Candida guiliermondii, Candida famata, Kodomala ohmeri, Trichosporon asahiu e Cryptococcus laurentii. Stagonosporopsis cucurbitacearum e leveduras pertencentes ao gênero Candida foram os microrganismos de maior ocorrência nas amostras da própolis provenientes da Baía do Iguape, Brasil.
- Enzimas β-Glucanases: aplicações da hidrólise de polissacarídeos /β-Glucanases enzymes – Appliance of polysaccharides hydrolisisPublication . Costa, Rodrigo Arthur Fonseca; Choupina, AltinoThe enzymatic degradation of polysaccharides is accomplished through the action of several hydrolases with different specificities and way of action. For this reason, there is now a growing interest in the study of these enzymes related to its potential industry. The β-glucanases enzymes belong to this group of hydrolases because of its ability to hydrolyze glycosidiclinkages β-D-glucosides 1,3-β-glucans and 1,6-β-glucans. Its applications are fairly comprehensive, with utility from conventional industry to the biotechnology and pharmaceutical industry.
- Knockdown of Phytophthora cinnamomi gip gene by iRNAPublication . Chahed, Abdessalem; Costa, Rodrigo Arthur Fonseca; Sousa, Maria João; Choupina, AltinoInk Disease is considered one of the most important causes of the decline of chestnut orchards. The break in yield of Castanea sativa Mill is mainly caused by Phytophthora cinnamomi one of the most aggressive and widespread plant pathogen causing enormous economic losses and up to now no efficient treatments are available to fight these pathogens. Because of the importance of chestnut at economical and ecological levels especially in Portugal, it becomes essential to explore the molecular mechanisms that determine the interaction between Phytophthora species and host plants through the study of GIP protein produced by P.cinnamomi during the infection.
- Post-transcriptional gene silencing of glucanase inhibitor protein in Phytophthora cinnamomiPublication . Ferreira, Patrick; Chahed, Abdessalem; Estevinho, Leticia M.; Seixas, Natália; Costa, Rodrigo Arthur Fonseca; Choupina, AltinoInk disease is considered one of the most significant causes contributing to the decline of chestnut orchards. The reduced yield of Castanea sativa Mill can be attributed to two main species: Phytophthora cinnamomi and Phytophthora cambivora, with the first being the main pathogen responsible for ink disease in Portugal. P. cinnamomi is a highly aggressive and widely distributed plant pathogen, capable of infecting nearly 1000 host species. This oomycete causes substantial economic losses and is accountable for the decline of numerous plant species in Europe and worldwide. To date, no effective treatments are available to combat these pathogens. Given chestnut’s economic and ecological significance, particularly in Portugal, it is crucial to investigate the molecular mechanisms underlying the interaction between Phytophthora species and host plants. This can be achieved through the study of the glucanase inhibitor protein (GIP) produced by P. cinnamomi during infection. The technique of RNA interference (RNAi) was employed to suppress the GIP gene of P. cinnamomi. The resulting transformants, carrying the silenced gene, were used to infect C. sativa, allowing for the assessment of the effects of gene silencing on the plant’s phenotype. Additionally, bioinformatics tools predicted the secretion of GIP protein. The obtained results validate RNAi as a potential alternative tool for studying molecular factors and for controlling and managing P. cinnamomi.
- Sequence analysis and expression of genes involved cytoskeleton and infection mechanisms in the plant pathogen Phytophthora cinnamomiPublication . Choupina, Altino; Estevinho, Leticia M.; Costa, Rodrigo Arthur Fonseca; Dominguez, ÁngelOomycetes from the genus Phytophthora are plant pathogens that are devastating for agriculture and natural ecosystems. The biggest productivity and yield break occurs due to the ink disease; caused by Phytophthora cinnamomi, which is one of the most widely distributed Phytophthora species, with nearly 1 000 host species. The knowledge about the molecular mechanisms responsible for its pathogenicity is an important tool in order to fight diseases associated with this pathogen.
- Silencing the gip gene of Phytophthora cinnamomi by iRNA and subcellular localization of GIP and NPP1 proteinsPublication . Chahed, Abdessalem; Chatti, Noureddine; Costa, Rodrigo Arthur Fonseca; Sousa, Maria João; Choupina, AltinoInk Disease is considered one of the most important causes of the decline of chestnut orchards. The break-in yield of Castanea sativa Mill is mainly caused by Phytophthora cinnamomi one of the most aggressive and widespread plant pathogen causing enormous economic losses and up to now, no efficient treatments are available to fight these pathogens. Because of the importance of chestnut at economical and ecological levels especially in Portugal, it becomes essential to explore the molecular mechanisms that determine the interaction between Phytophthora species and host plants through the study of proteins GIP and NPP1 produced by P.cinnamomi during the infection.
- Silencing the gip gene of Phytophthora cinnamomi by iRNA and subcellular localization of GIP and NPP1 proteinsPublication . Chahed, Abdessalem; Chatti, Noureddine; Costa, Rodrigo Arthur Fonseca; Sousa, Maria João; Choupina, AltinoInk Disease is considered one of the most important causes of the decline of chestnut orchards. The break in yield of Castanea sativa Mill is mainly caused by Phytophthora cinnamomi one of the most aggressive and widespread plant pathogen causing enormous economic losses and up to now no efficient treatments are available to fight these pathogens. Because of the importance of chestnut at economical and ecological levels especially in Portugal, it becomes essential to explore the molecular mechanisms that determine the interaction between Phytophthora species and host plants through the study of proteins GIP and NPP1 produced by P.cinnamomi during the infection.