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- A Reliable Molecular Diagnostic Tool for CA90 (Castanea sativa × Castanea crenata) Hybrid Identification Through SSRPublication . Yussif, Toufiq Soale; Cruz, Nadine Evora da; Coelho, Valentim; Gouveia, Maria Eugénia; Choupina, AltinoChestnut trees are an essential source of both food and timber. However, the severe threats from invasive pests and diseases compromise their existence and productivity. In Europe, chestnut hybridization programs have been initiated to produce resilient rootstocks in response to ink disease. However, the gap in the identification of these hybrid plants is typically based on field observations and Morphological features and remains a challenge. Our study presents a marker set for distinguishing between chestnut hybrid CA90 (Castanea sativa × Castanea crenata), a hybrid with demonstrated resistance to Phytophthora cinnamomi, and other varieties using microsatellite (SSR) markers and bioinformatics tools. We used 35 chestnut samples, including three CA90 controls, hybrids sampled within Portugal, with an aim to define the profiles of the chestnut hybrids and varieties in this study based on band patterns and SSR motifs. We selected and modified nine distinct SSR primers with null allelic features from 43 already developed simple sequence repeat (SSR) markers. PCR amplification and agarose gel electrophoresis were used to amplify and visualize the DNA bands. To confirm genetic variations, 27 amplified bands were sequenced by Sanger sequencing. This analysis identified 31 SSRs across 22 SSR-containing sequences, with trinucleotide (67.74%) repeats being the most common, followed by repeats of dinucleotide (22.58%), mononucleotide (6.45%), and hexanucleotide (3.23%). A total of 18 alleles were observed for the nine loci. The alleles ranged from one to three per locus for the 35 samples. The novel locus CP4 could only be found in CA90 hybrids. This tool can aid in identifying and selecting disease-resistant hybrids, thereby contributing to chestnut production and management strategies.
- 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.
- Isolation and sequence analysis of alfa-tubulin gene from Phytophora cinnamomiPublication . Dias, Teresa; Andrade, Maria; Jorge, Lurdes; Vaz, Madalena; Martins, Fátima; Dominguez, Ángel; Choupina, AltinoPhytophthora diseases cause widespread economic and environmental losses worldwide. Thousands of plant species are susceptible. In Portugal, Phytophthora cinnamomi is responsible for chestnut ink disease. Despite the differences there are a number of key steps common to most infection strategies, including adhesion to the plant surface, plant penetration through the secretion of a diverse range of cell wall-degrading enzymes and hyphal growth. The cell cytoskeleton plays a critical role in these processes. Microtubules are a major constituent of the cell cytoskeleton. They participate in a wide range of cellular functions, such as motility, division, maintenance of cell shape, and intracellular transport. However, microtubule role is variable depending on the organism, cell type and other factors. Tubulin is the major constituent of microtubules and is composed of a heterodimer of two closely related proteins, alpha and beta tubulin. In S. cerevisiae cells, the essential TUB1 gene is the major gene, while the nonessential gene TUB3 is a minor gene, encoding α-tubulin. The β-tubulin subunit is encoded by the TUB2 gene. In Magnaporthe grisea both α-and β-tubulins are found as single-copy genes. The Oomycetes are, however, phylogenetically quite distinct from the fungi. Analysis of structural, biochemical and molecular characteristics have led to the Oomycetes being grouped with the chromophyte algae. In order to elucidated the role of cytoskeleton in pathogenicity mechanisms of Phytophthora cinnamomi, was cloned a gene encoding alpha-tubulin from P. cinnamomi. To isolated this gene, the existing Tub1 nucleotide sequences were retrieved from the NCBI GenBank (www.ncbi.nlm.nih.gov/genbank). These sequences were aligned in Clustal and degenerate primers Tub1 and Tub2 were designed. A 1200bp fragment was generated from genomic DNA by PCR and subsequently cloned into pGEM-T vector. To complete the open reading frame it was used the HE-TAIL PCR. The complete ORF was sequenced and submitted in EMBL databases (Accession number AM412177.1). Based on the computational analysis through BioEdit software, TUB1 has a 1362 bp ORF and encodes a 453 a.a protein with a molecular weight of 49,911kDa. Phylogenetic analysis of deduced amino acid sequence using FASTA programs from EMBL databases revealed that Tub1 revealed 99.6% identity with alpha-tubulin of P. infestans T30.4 and 98.9% identity with P. capsici, but only 68,1 % with alpha-tubulin of S. cerevisiae.
- 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.
- Expression analysis by RT-PCR of GIP gene from Phytophthora cinnamomiPublication . Belo, Hélio; Martins, Fátima; Jorge, Lurdes; Sousa, Maria João; Rodrigues, Luciano; Choupina, AltinoSpecies of the genus Phytophthora secrete glucanase inhibitor proteins (GIPs) to inhibit the activity of enzymes involved in plant defense responses, including during plant infection process of Castanea sativa Mill by Phytophthora cinnamomi. GIPs show structural homology to the chymotrypsin class of serine proteases (SP) but lack proteolytic activity due to the absence of an intact catalytic triad and, thus, belong to a broader class of proteins called serine protease homologs (SPH) nonfunctional because one or more residues of the essential catalytic triad is absent (His-Asp-Ser). GIPs show high homology to the S1A subfamily of SP, however questions remain about the expression patterns and potential roles of different GIPs during pathogenesis and their possible interaction with host EGases in the plant apoplast. ORF of GIP gene from P. cinnamomi encodes a 269 aa protein. In order to understand its function, we proceeded to the heterologous expression in Pichia pastoris. The expression was studied during growth in different carbon sources and a time course of glucanase inhibitor protein production by RT-PCR was also performed. The major expression levels occurred at the medium with glucose as carbon source.
- 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.