Browsing by Author "Coelho, A. C."
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- A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencingPublication . Pereira-Leal, José B.; Abreu, Isabel A.; Alabaça, Cláudia S.; Almeida, Maria H.; Almeida, Paulo; Almeida, Tânia; Amorim, Maria I.; Araújo, Susana; Azevedo, Herlânder; Badia, Aleix; Batista, Dora; Bohn, Andreas; Capote, Tiago; Carrasquinho, Isabel; Chaves, Inês; Coelho, A. C.; Costa, Maria M. R.; Costa, Rita; Cravador, A.; Egas, Conceição; Faro, Carlos; Fortes, Ana M.; Fortunato, Ana S.; Gaspar, Maria J.; Gonçalves, Sónia; Graça, José; Horta, Marília; Inácio, Vera; Leitão, J. M.; Lino-Neto, Teresa; Marum, Liliana; Matos, José; Mendonça, Diogo; Miguel, Andreia; Miguel, Célia M.; Morais-Cecílio, Leonor; Neves, Isabel; Nóbrega, Filomena; Oliveira, Maria M.; Oliveira, Rute; Pais, Maria S.; Paiva, Jorge A.; Paulo, O. S.; Pinheiro, Miguel; Raimundo, João A. P.; Ramalho, J. C.; Ribeiro, Ana I.; Ribeiro, Teresa; Rocheta, Margarida; Rodrigues, Ana I.; Rodrigues, José C.; Saibo, Nelson J. M.; Santo, Tatiana; Santos, Ana M.; Sá-Pereira, Paula; Sebastiana, Mónica; Simões, Fernanda; Sobral, Rómulo S.; Tavares, Rui; Teixeira, Rita; Varela, Carolina; Veloso, Maria M.; Ricardo, Cândido P. P.Background: Cork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management. Results: We generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org. Conclusions: This genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.
- Genetic diversity of two evergreen oaks (Quercus suber L. and Q (ilex) rotundifolia Lam.) in Portugal using AFLP markersPublication . Coelho, A. C.; Lima, M. B.; Neves, D.; Cravador, A.The genetic variability of cork oak (Quercus suber, L.) in Portugal was evaluated by AFLP using five primer combinations. Three hundred and thirteen trees from three geographically contrasting regions exhibited a high level of genetic variation. The genetic profile of each individual is composed of 291 loci, randomly positioned in the genome and consists of monomorphic and polymorphic fragments. Similarities and dissimilarities among the individuals were quantitatively evaluated by numerical taxonomy. The overall sample shows a proportion of AFLP polymorphic markers of 71%, denoting a high level of variability. Ninety percent of the polymorphic markers identified in cork oak genotypes are uniformly distributed throughout the cork oak populations of Algarve, Alentejo and Trás-os-Montes regions. The coefficients of genetic similarity vary from 0.61 to 0.88 implying that 60% of fragments found are common. A sample of 52 holm oak [Quercus ilex subsp. rotundifolia (Lam.)] trees from overlapping areas was also analysed by AFLP with the same five primer combinations. However the codification of markers together with those selected on cork oak profiles was feasible with only one primer combination due to an apparent much higher polymorphism. AFLP and numerical taxonomy analysis enabled to differentiate the taxa and showed that the level of similarity observed between the profiles of the individuals from holm oak species was lower than that observed in cork oak, implying that apparently the degree of polymorphism is higher in Q. ilex subsp. rotundifolia than that quantified in Q. suber. A Bayesian approach was used to assess Q. suber total genetic diversity (Ht = 0.2534, P < 0.001) of which 1.7% (Fst = 0.0172, P < 0.001) was assigned to differences among populations. Analysis of molecular variance (AMOVA) showed that most genetic variation is comprised within populations (96%) while 3.6% is among populations (Φst = 0.036, P < 0.001). Differences among populations within geographic regions account for 2.6% (Φsc = 0.026, P < 0.001) of the total variation and only 1.3% (Φct = 0.013, P = 0.007) is attributed to variation among regions denoting little differentiation of populations over a range of 700 km.
- Highly specific and sensitive non-radioactive molecular identification of Phytophthora cinnamomiPublication . Coelho, A. C.; Cravador, A.; Bollen, A.; Ferraz, J. F. P.; Moreira, A. C.; Fauconnier, A.; Godfroid, EdmondIn response to the need for a faster, more reliable method for identifying Phytophthora cinnamomi in cork oak soils in Portugal, a simple, fast, sensitive molecular identification method is described. It is based on a colorimetric assay which involves an oligonucleotide capture probe covalently immobilised on microtitration wells, a multi-biotinylated oligonucleotide detection probe and the PCR-amplified target DNA. The target DNA is a 349 bp DNA fragment partially covering the 3'-translated and 3'- untranslated regions of the cinnamomin gene. When the specificity of the PCR reaction was evaluated in vitro using isolates of P. cinnamomi and eight other Phytophthora species, including the related P. cambivora, it was specific to P. cinnamomi. When 30 isolates of P. cinnamomi from oak roots in southern Portugal were assayed, 26 gave a strong positive response. The assay has a sensitivity of about 2±5 genome equivalents of P. cinnamomi. The reason for the negative response of four isolates remains unclear.
- Identification of an elicitin gene cluster in Phytophthora cinnamomiPublication . Duclos, J.; Fauconnier, A.; Coelho, A. C.; Bollen, A.; Cravador, A.; Godfroid, EdmondElicitins are a group of highly conserved proteins secreted by species of Phytophthora and a species of the related genus Pythium, Pythium vexans. Some of these proteins act as inducers of the necrotic hypersensitive-like response and the associated systemic acquired resistance phenomenon, in some species. We cloned and characterised the cinnamomin-beta and -alpha genes and two related elicitin genes from Phytophthora cinnamomi. These four open reading frames (ORFs) are clustered in tandem pairs. Two out of these four genes present homologies with the basic and acidic elicitin groups; but the two others encode, if expressed, elicitin isoforms exhibiting homologies with the class II of highly acidic elicitins.
- Identification of an elicitin gene cluster in Phytophthora cinnamomi and analysis of the necrotic activity of a purified recombinant beta-cinnamominPublication . Duclos, J.; Aurélio, M.; Graca, J.; Coelho, A. C.; Fauconnier, A.; Jacquet, Alain; Bollen, A.; Cravador, A.; Biemans, R.; Godfroid, EdmondThe oomycetous fungus Phytophthora cinnamoni plays and important role in the necrotic activity observed on feeder root of cork oaks (Quercus suber L.) and eucalyptus (eucalyptus marginata Donn. Ex. Sm.)trees reducing the capacity of these hosts to absorb water and nutrients.
- In vitro and in vivo quantification of elicitin expression in Phytophthora cinnamomiPublication . Horta, Marília; Sousa, Nelson; Coelho, A. C.; Neves, D.; Cravador, A.The differential expression of four Phytophthora cinnamomi elicitin genes was analysed by Real Time RT-PCR. In in vitro cultures, the a-cinnamomin gene showed the highest level of expression, the b-cinnamomin gene (b-cin) was the most inducible, and the HAE transcripts were in low abundance. Transcription of all the elicitins was active during the active growth of the pathogen when infecting cork oak (Quercus suber) roots, and as host colonization progressed, the level of b-cin expression fell, while that of a-cin rose. In an antisense transgenic strain, the silencing of b-cin also negatively affected the expression of other elicitin genes in the cluster. The reduced in planta growth of the b-cin knock-out is related to the altered pattern of elicitin gene expression, supporting the idea that one of the functions of elicitins is related, directly or indirectly, with pathogenesis.
- Involvement of a cinnamyl alcohol dehydrogenase of Quercus suber in the defence response to infection by Phytophthora cinnamomiPublication . Coelho, A. C.; Horta, Marília; Neves, D.; Cravador, A.A gene encoding a potential NADPH-dependent cinnamyl alcohol dehydrogenase (QsCAD1) (GenBank accession no: AY362455) was identified in Quercus suber (cork oak). Its complete cDNA sequence was obtained by RACE-PCR, starting from total RNA extracted from roots of seedlings of Q. suber, infected with Phytophthora cinnamomi, the causal agent of the decline and sudden death of Q. suber and Quercus ilex subsp. rotundifolia in the Iberian Peninsula. Sequence information to perform the RACE-PCR was acquired from a polymorphic fragment (C9), specifically identified by cDNA-AFLP, in leaves of epicormic shoots of a cork oak tree that suffered sudden death. RT-PCR and hybridization analysis showed that the QsCAD1 gene is up-regulated in root seedlings of Q. suber infected with P. cinnamomi. QsCAD1 has a high structural homology with VR-ERE (Vigna radiata), an enzyme that detoxifies eutypine (produced by Eutypa lata, the causal agent of eutypa dieback of grapevines), to eutypinol, and with QrCAD1 (Q. ilex subsp. rotundifolia), EgCAD1 (Eucalyptus gunnii), MdCAD1 (Malus x domestica). Taken together, these results suggest that these enzymes, and namely QsCAD1 belong to a new group of CAD potentially involved in deactivation of toxins produced by phytopathogens.
- Quercus suber – P. cinnamomi interaction: hypothetical molecular mechanism modelPublication . Coelho, A. C.; Horta, Marília; Ebadzad, G.; Cravador, A.Phytophthora cinnamomi Rands is involved in the decline and mortality of Quercus suber L. and Quercus ilex L. in Southern Europe, in particular in Portugal and Spain. The presence and spread of P. cinnamomi in these regions is a severe threat to these oak ecosystems leading to expectable severe consequences for the production of cork and acorns in the near future. Molecular mechanisms underlying oomycete-host interactions are poorly understood. As a first step to identify transcripts involved in the Quercus suber – Phytophthora cinnamomi interaction, we applied complementary deoxyribonucleic acidamplified fragment length polymorphism (cDNA-AFLP) methodology to cork oak seedlings infected with zoospores or mycelium of P. cinnamomi. Forty-four Quercus suber genes that were differentially expressed when exposed to Phytophthora cinnamomi were selected and sequenced. Several of these genes were fully sequenced and the deduced aminoacid sequences showed consistent homology with proteins involved in the defence mechanism of other plant species. These findings led to the design of a simplified hypothetical model that illustrates the initial events of the interaction between Q. suber and P. cinnamomi.
- Strategies of attack and defence in woody plant-Phytophthora interactionsPublication . Oßwald, W.; Fleischmann, F.; Rigling, D.; Coelho, A. C.; Cravador, A.; Diez, J.; Dalio, R. J.; Horta Jung, Marília; Pfanz, H.; Robin, C.; Sipos, G.; Solla, A.; Cech, T.; Chambery, A.; Diamandis, S.; Hansen, E.; Jung, Thomas; Orlikowski, L. B.; Parke, J.; Prospero, S.; Werres, S.; Vannini, A.This review comprises both well-known and recently described Phytophthora species and concentrates on Phytophthora–woody plant interactions. First, comprehensive data on infection strategies are presented which were the basis for three models that explain invasion and spread of Phytophthora pathogens in different woody host plants. The first model describes infection of roots, the second concentrates on invasion of the trunk, and the last one summarizes infection and invasion of host plants via leaves. On the basis of morphological, physiological, biochemical and molecular data, scenarios are suggested which explain the sequences of reactions that occur in susceptible and tolerant plants following infections of roots or of stem bark. Particular emphasis is paid to the significance of Phytophthora elicitins for such host–pathogen interactions. The overall goal is to shed light on the sequences of pathogenesis to better understand how Phytophthora pathogens harm their host plants.
- Temporal metabolic profiling of the Quercus suber-phytophthora cinnamomi system by middle-infrared spectroscopyPublication . Hardoim, P.R.; Guerra, Rui Manuel Farinha das Neves; Rosa Da Costa, Ana; Serrano, M. S.; Sanchez, M. E.; Coelho, A. C.The oomycete Phytophthora cinnamomi is an aggressive plant pathogen, detrimental to many ecosystems including cork oak (Quercus suber) stands, and can inflict great losses in one of the greatest hotspots' for biodiversity in the world. Here, we applied Fourier transform-infrared (FT-IR) spectroscopy combined with chemometrics to disclose the metabolic patterns of cork oak roots and P.cinnamomi mycelium during the early hours of the interaction. As early as 2h post-inoculation (hpi), cork oak roots showed altered metabolic patterns with significant variations for regions associated with carbohydrate, glycoconjugate and lipid groups when compared to mock-inoculated plants. These variations were further extended at 8hpi. Surprisingly, at 16hpi, the metabolic changes in inoculated and mock-inoculated plants were similar, and at 24hpi, the metabolic patterns of the regions mentioned above were inverted when compared to samples collected at 8hpi. Principal component analysis of the FT-IR spectra confirmed that the metabolic patterns of inoculated cork oak roots could be readily distinguished from those of mock-inoculated plants at 2, 8 and 24hpi, but not at 16hpi. FT-IR spectral analysis from mycelium of P.cinnamomi exposed to cork oak root exudates revealed contrasting variations for regions associated with protein groups at 16 and 24h post-exposure (hpe), whereas carbohydrate and glycoconjugate groups varied mainly at 24hpe. Our results revealed early alterations in the metabolic patterns of the host plant when interacting with the biotrophic pathogen. In addition, the FT-IR technique can be successfully applied to discriminate infected cork oak plants from mock-inoculated plants, although these differences were dynamic with time. To a lesser extent, the metabolic patterns of P.cinnamomi were also altered when exposed to cork oak root exudates.