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- 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.
- 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.
- Análise bioinformática da estrutura e função da informação biológicaPublication . Choupina, Altino; Deusdado, SérgioO conhecimento derivado das tecnologias genómicas e computacionais aumenta em progressão geométrica. A compreensão dessa avalanche de dados está intimamente vinculada ao formidável desenvolvimento na área da bioinformática. Ao possibilitar a avaliação global dessa extraordinária quantidade de dados, a bioinformática tem acelerado consideravelmente as descobertas científicas. No software de análise do genoma podem encontrar-se diversos pacotes de programas, os quais acompanham todo o processo desde a receção dos gráficos provenientes do sequenciador até à publicação dos dados em bases de dados on-line. Estas características, juntamente com o acesso grátis para académicos, a compatibilidade de ficheiros, e a sua data de conceção são os principais fatores de seleção nas escolhas realizadas.
- 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.
- Estudo do comportamento de leveduras isoladas de melPublication . Calhelha, Ricardo C.; Estevinho, Isabel; Barbosa, Sandra; Choupina, Altino; Estevinho, Leticia M.Foram isoladas leveduras em amostras de me! e identificados através de métodos tradicionais e métodos moleculares. Nos métodos moleculares foram utilizados os primers NL1 e NL2 com os quais se amplificou a região D1/D2 do gene 26 rRNA.
- Bioinformatics tools for identification of pathogenic factorsPublication . Choupina, AltinoThe culture of the chestnut tree is extremely important in the northern region of Portugal, occupying a significant! proportion of useful agricultural area. The annual average chestnut production in Portugal can reach 20 000 tons. New plantation areas have increased in the last law decades. However the ink disease caused by the oomycete Phytophthora cinnamomi has damage and killed many trees and up to now no concrete solutions have been offered to control the illness. As a consequence, the disease propagation in the orchards 01 chestnut trees has been causing severe productivity and yield breaks. ln addition to the economical lasses, the importance of sociological and landscape aspects for the region cannot be neglected
- Valorização de produtos na produção de extrudidosPublication . Choupina, Altino; Lopes-da-Silva, M.F.; Santos, Luís; Costa, Luísa Beirão daA extrusão é uma etapa de processamento de matéria-prima sólida que junta num único equipamento, denominado extrusora, várias operações unitárias e modificações físico-químicas. Ensaiou-se a produção de produtos extrudidos a partir de castanha de menor valor comercial numa extrusora de parafuso simples, fazendo variar parâmetros como a velocidade do parafuso. Avaliaram-se comparativamente as características dos produtos obtidos nas diversas combinações das farinhas de milho e de castanha, nas múltiplas condições ensaiadas, através das modificações funcionais, por microscopia ótica e pelo comportamento amilográfico, e das alterações a nível molecular, por cromatografia de filtração sobre gel. Foi também determinado o respetivo perfil instrumental de textura, assim como foi realizada uma avaliação sensorial dos produtos obtidos. Os resultados mostraram que o entumescimento dos grânulos de amido, a relação amilose/amilopectina, a massa molecular da amilopectina e o teor em lípidos da castanha são muito superiores às do milho, justificando as diferenças encontradas nas respectivas curvas de consistência e as alterações sofridas na extrusão. Os produtos com maior aceitabilidade foram os processados com baixos teores de humidade e temperaturas de extrusão intermédias.
- Lipases: biocatalizadores da hidrólise de triacilgliceroisPublication . Vaz, Madalena; Choupina, AltinoOs enzimas lipases (E.C.3.1.1.3) pertencem à família das serinas hidrolases, que por sua vez incluem várias enzimas esterases (E.C.3.1.1.1). Uma característica que distingue as lipases das esterases é o facto de serem capazes de realizar a hidrólise de óleos e gorduras em glicerol e ácidos gordos livres na interface óleo-água. Normalmente é aceite que as esterases hidrolisam ligações éster em esteres e triacilgliceróis de cadeia carbónica curta, libertando ácidos gordos de baixo peso molecular, já as lipases hidrolisam preferencialmente triacilglicerois de cadeia longa. As lipases microbianas apresentam uma grande diversidade de aplicações industriais tais como na indústria farmacêutica, alimentar, de detergentes entre outros.
- In silico characterization of molecular factors involved in metabolism and pathogenicity of Phytophthora cinnamomiPublication . Boughanmi, Mohamed Belhassan; Branco, Iuliia; Choupina, AltinoPhytophthora cinnamomi is classified as one of the most devastating plant pathogens in the world. It has a destructive effect on more than 5000 horticultural and forestry species in the world, and especially on Castanea sativa. The genus Phytophthora belongs to the Class Oomycetes, a group of fungus like organisms which provoke plant diseases via motile zoospores. Control of this organism is considered very challenging because of the limited range of effective chemical inhibitors. The development of sustainable control measures for the future management of P. cinnamomi requires in-depth knowledge of the cellular and molecular bases of development and metabolism. The aim of this review was to identify molecular factors associated with the metabolism of P. cinnamomi by studying the genes implicated in fundamental metabolism using tools of bioinformatics. Also, some genes involved in pathogenicity will be cited and characterized, such as genes coding for transglycosylases. Genomic sequences of P. cinnamomi were analyzed using an open reading frame (ORF) finder. The identified ORFs products (proteins) were compared to sequences already described and with known functions present in databases such as NCBI and fungi database. In this way, homologous proteins were found, with the respective specific domains, to proteins involved in the metabolism and pathogenicity of Phytophthora ssp.
- Biochemical and molecular classification of yeasts from Trás-os-Montes honeyPublication . Carvalho, Marisa; Pinhel, Isabel; Rocha, Amélia; Choupina, Altino; Estevinho, Leticia M.Anexo