Browsing by Author "Martins, Ivone"
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- Bioactive properties and functional constituents of Hypericum androsaemum L.: A focus on the phenolic profilePublication . Jabeur, Inès; Tobaldini, Flávia; Martins, Natália; Barros, Lillian; Martins, Ivone; Calhelha, Ricardo C.; Henriques, Mariana; Silva, Sónia; Achour, Lotfi; Santos-Buelga, Celestino; Ferreira, Isabel C.F.R.Hypericum androsaemum L. ethanol:water extract acted as a lipid peroxidation inhibitor and free radical scavenger. Amarked inhibition of the growth of breast, lung, cervical and hepatocellular human carcinoma cell lineswas also observed, whereas no toxicity was shown against non-tumor porcine liver cells (N400 μg/mL). The extract was also effective in inhibiting nitric oxide production, as an indicator of the anti-inflammatory potential. The anti-Candida effects varied among different strains of the same species, C. glabrata and C. tropicalis being the most sensible species with an effect directly related with the extract concentrations tested. A significant antibiofilm formation potential was also observed, namely for C. glabrata and C. tropicalis (biofilm reduction N90%). 5-O-Caffeoylquinic and 3-O-caffeoylquinic acids were themost abundant phenolic compounds identified in the extract, and might be related with the observed bioactive effects. Nevertheless, future studies should be carried out to obtain dose-response curves of the isolated active compounds, in order to perform further preclinically testing to quantify the presence of the most active compounds in the extract.
- Characterization of transglutaminase elicitor precursor from plants pathogen Phytophthora cinnamomiPublication . Carvalho, Marisa; Martins, Ivone; Meirinho, Sofia G.; Belo, Hélio; Choupina, AltinoThe oomycetes form a phylogenetically distinct group of eukaryotic microorganisms that includes some of the most notorious pathogens of plants. Among these, members of the genus Phytophthora cause enormous economic losses on crop species as well as environmental damage in natural ecosystems. Phytophthora cinnamomi is the most widely distributed Phytophthora species, with nearly 1000 host species.
- Cloning and expression analysis of glucanase genes from Phytophthora cinnamomiPublication . Martins, Ivone; Meirinho, Sofia G.; Dias, Teresa; Jorge, Lurdes; Martins, Fátima; Choupina, AltinoPhytophthora cinnamomi is one among the most destructive species of Phytophthora associated to the decline of forestry, ornamental and fruit species. Associated with this oomycete is the ink disease of Castanea sativa Mill. Glucan endo-1,3-β-D-glucosidase catalyzes the hydrolysis of 1,3-β-D-glucoside linkages in callose, laminarin and several carbohydrates found in the cell wall of plants and fungi. It is generally thought that glucanases play a role in plant defence by digesting wall components of the fungal pathogen. In oomycetes, glucanases have been studied at biochemical level for their possible role in hyphal tip growth and branching, where there is thought to be a delicate balance between the cell wall synthesis and hydrolysis. Fungal cell wall degrading enzyme production is influenced by a number of factors including the type of strain, the culture conditions and substrate type. The aim of this work was the analysis of homologous expression, in P. cinnamomi, and heterologous expression, in Pichia pastoris, of the endo-1,3-β-D-glucosidase encoding gene ENDO1 produced by P. cinnamomi. The expression was studied during growth in different carbon sources and was also performed a time course of endo-1,3-β-D-glucosidase production. Different plasmids were used to clone the gene on each organism and we used RT-PCR analysis to examine its expression. The major expression levels occurred at the medium with glucose as carbon source. These and other results will be presented.
- Cloning and expression analysis of glucanase genes from Phytophthora cinnamomiPublication . Martins, Ivone; Meirinho, Sofia G.; Dias, Teresa; Jorge, Lurdes; Martins, Fátima; Choupina, AltinoPhytophthora cinnamomi is a soil-borne pseudofungus belonging to the Class Oomycetes or „water moulds' in the Kingdom Chromista (Figure 1). Is one among the most destructive species of Phytophthora associated to the decline of forestry, ornamental and fruit species, as well as of some 900 other woody perennial plant species Associated with this oomycete is the ink disease of Castanea Sativa Mill. Glucan endo-1,3-β-D-glucosidase (EC 3.2.1.39) catalyzes de hydrolysis of 1,3-β-D-glucoside linkages in callose, laminarin and several carbohydrates found in the cell wall of plants and fungi. It is generally thought that glucanases play a role in plant defense by digesting wall components of the fungal pathogen. In yeast, 1,3-- glucanases have been studied for their role in germination, sporulation, mating and cell growth since they are regulated in cell cycle dependent manner, and are differentially expressed during vegetative growth, mating and the late stages of sporulating diploid. In plant, 1,3--glucanases have been characterized for their major role in plant defence, as well as for their involvement in germination, microsporogenesis and embryogenesis. In oomycetes, glucanases have been studied on a biochemical level for their possible role in hyphal tip growth and branching where there is thought to be a delicate balance between cell wall synthesis and hydrolyses. In the present work, we obtained a fragment with 1231bp of the endo-1,3--glucanase gene by standard PCR, using conserved primers and the whole genomic sequence with 2586 bp was obtained by amplifying the previous sequence by asymmetric PCR. The gene expression was studied during growth in different carbon sources and was also performed a time course of endo-1,3-β-D-glucosidase production.
- Cloning, characterization and in vitro and in planta expression of a glucanase inhibitor protein (GIP) of Phytophthora cinnamomiPublication . Martins, Ivone; Martins, Fátima; Belo, Hélio; Vaz, Madalena; Carvalho, Marisa; Cravador, Alfredo; Choupina, AltinoOomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. They are able to secrete a glucanase inhibitor protein (GIP) that inhibits the activity of endoglucanases (EGases) involved in defense responses against infection. One of the most widely distributed and aggressive Phytophthora species, with more than 1,000 host plants is P. cinnamomi. In this work we report the sequencing and characterization of a class of GIPs secreted by Phytophthora cinnamomi. The gip gene from P. cinnamomi has a 937 bp ORF encoding a putative peptide of 312 deduced amino acids. The expression of this gene was studied during growth in different carbon sources (glucose, cellulose and sawdust), by RT-qPCR and its level of expression was evaluated at five time points. The highest expression of gip gene occurred in sawdust at 8 h of induction. In vivo infection of C. sativa revealed an increase in gip expression from 12 to 24 h. At 36 h its expression decreased suggesting that a compensatory mechanism must occur in plant.
- 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.
- Genes found in partial sequencing of Phytophthora cinnamomiPublication . Santos, Luís; Martins, Ivone; Maia, Vera; Dominguez, Ángel; Choupina, AltinoMembers of the oomycete cause extensive losses in agriculture and widespread degradation in natural plant communities, being responsible for the death of thousands of trees every year. Two of the representative species are Phytophthora infestans, which causes late blight of potato, and Phytophthora cinnamomi, which causes chestnut ink disease, responsible for losses on sweet chestnut production in Europe. Genome sequencing efforts have been focused on the study of three species: P. infestans, P. sojae and P. ramorum. Phytophthora infestans has been developed as the model specie for the genus, possessing excellent genetic and genomics resources including genetic maps, BAC libraries, and EST sequences. Our research team is trying to sequence the genome of P. cinnamomi in order to gain a better understanding of this oomycete, to study changes in plant-pathogen relationships including those resulting from climate change and trying to decrease the pathogen’s impact on crops and plants in natural ecosystems worldwide. We present here a preliminary report of partially sequenced genomic DNA from P. cinnamomi encoding putative protein-coding sequences and tRNAs. Database analysis reveals the presence of genes conserved in oomycetes.
- Glucanase Inhibitor Protein (GIP)Publication . Choupina, Altino; Martins, IvoneIn order to inhibit the activity of plant extracellular enzymes, β(1,3) and β(1,6)endoglucanases (EGases), involved in plant defense responses, including during the infection process, glucanase inhibitor proteins (GIPs) are secreted by species of the genus Phytophthora. Besides the structural homology to the chymotrypsin class of serine proteases (SP), GIPs, due to the absence of an intact catalytic triad, lack proteolytic activity. For that reason, they belong to a broader class of nonfunctional proteins caled serine protease homologs (SPH). Despite the high homology of GIPs to the SP subfamily S1A, some questions remain regarding the expression patterns and potential roles of different GIPs during pathogenesis and also their possible interaction with host EGases in the plant apoplast.
- Isolation and characterization of necrosis-inducing phytophthora protein 1 (npp1) gene from plants pathogen Phytophthora cinnamomiPublication . Martins, Ivone; Meirinho, Sofia G.; Belo, Hélio; Vaz, Madalena; Martins, Fátima; Choupina, AltinoOomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. Due to their particular physiological characteristics, no efficient treatments against diseases caused by these microorganisms are presently available. To develop such treatments, it appears essential to dissect the molecular mechanisms that determine the interaction between Phytophthora species and host plants. One of the most widely distributed Phytophthora specie, with nearly 1000 host species is Phytophthora cinnamomi. Associated with this pathogen is the ink disease of Castanea Sativa Mill being one of the most destructive diseases in C. Sativa in the northeast of Portugal and the most common symptoms are root necrosis and reduction in root growth, which invariably lead to the trees death. P. cinnamomi is able to secrete a novel class of necrosis-inducing proteins, known as Nep1-like proteins (NLPs), more specifically necrosis-inducing Phytophthora protein 1 (npp1), that causes necrosis on leaf and roots of the plant, leading to the plant death. In order to better evaluate the mechanism of plant necrosis induced by P. cinnamomi. The study of factors that affect npp1 gene expression is extremely important. The npp1 gene ORF comprises 770 bp encoding a 256 aa protein with a molecular weight of approximately 25 kD. In order to understand its function, gene expression in vitro in P. pastoris (heterologous expression), was studied during growth in different carbon sources, by RT-qPCR. Over expression of our gene in P. pastoris was also performed. In vivo expression technology has been used to study the expression of npp1 gene from fungi during infection by RT-PCR. In our work chestnut roots were infected with P. cinnamomi and mRNA was extracted at different times of infection to analyze gene expression. These and other results will be presented and discussed.
- Isolation and Sequencing of Actin1, Actin2 and Tubulin1 Genes Involved in Cytoskeleton Formation in Phytophthora cinnamomiPublication . Martins, Ivone; Lopez, Carmen; Dominguez, Ángel; Choupina, AltinoOomycetes from the genus Phytophthora are fungus-like plant pathogens that are devastating for agriculture and natural ecosystems. On the Nordeste Transmontano region (northeast Portugal), the Castanea sativa chestnut culture is extremely important. 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 1000 host species. The knowledge about molecular mechanisms responsible for pathogenicity is an important tool in order to combat associate diseases of this pathogen. Complete open reading frames (ORFs) of act1, act2 and tub1 genes who participate in cytoskeleton formation in P. cinnamomi were achieved by high-efficiency thermal asymmetric interlaced (HE-TAIL) polymerase chain reaction (PCR). act1 gene comprises a 1128 bp ORF, encoding a deduced protein of 375 amino acids (aa) and 41,972 kDa. act2 ORF comprises 1083 bp and encodes a deduced protein of 360 aa and 40,237 kDa. tub1 has a total length of 2263 bp and encodes a 453 aa protein with a molecular weight of 49.911 kDa. Bioinformatics analyses shows that actin1 is ortholog to the act1 genes of Phytophthora infestans, Phytophthora megasperma and Phytophthora melonis; actin2 is ortholog to the act2 genes of P. infestans, Phytophthora brassicae, P. melonis and Pythium splendens and tubulin1 shows the highest orthology to P. infestans and P. capsici α-tubulin genes. Analysed 3D structure of the three putative proteins revealed a spatial conformation highly similar to those described for orthologous proteins obtained by X-ray diffraction.