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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.
- 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.
- 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.
- 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.
- Involvement of the beta-cinnamomin elicitin in infection and colonisation of cork oak roots by Phytophthora cinnamomiPublication . Cravador, A.; Horta, Marília; Caetano, P.; Medeira, C.; Maia, I.The virulence of two wild type (PA45 and PA37) and two genetically modified (13C: hygromycin resistant; FATSS: hygromycin resistant and β-cin knock-down) Phytophthora cinnamomi strains towards cork oak (Quercus suber) was assessed via a quantitative evaluation of disease symptoms arising from a soil infestation assay, and by a istological analysis of root colonization. Comparison of virulence, as expressed by symptom severity, resulted in the following ranking: highly virulent (wild type strains), medium virulence (strain 13C) and weakly virulent (FATSS). Both transgenic strains were compromised in their virulence, as expressed by symptom severity, but strain 13C was much less affected than FATSS. Microscopic observation showed that the FATSS strain was unable to effectively invade the root, while 13C and the two wild type strains were all able to rapidly colonize the whole root, including the vascular tissue. These results strengthen the notion that elicitins are associated, either directly or indirectly, with the infection process of Phytophthora.
- Nothophytophthora gen. nov., a new sister genus of Phytophthora from natural and semi-natural ecosystemPublication . Jung, Thomas; Scanu, B.; Bakonyi, J.; Seress, D.; Kovács, G.M.; Durán, A.; Sanfuentes von Stowasser, E.; Schena, L.; Mosca, S.; Thu, P.Q.; Nguyen, C.M.; Fajardo, S.; González, M.; Pérez-Sierra, A.; Rees, H.; Cravador, A.; Maia, Cristiana; Horta Jung, MaríliaDuring various surveys of Phytophthora diversity in Europe, Chile and Vietnam slow growing oomycete isolates were obtained from rhizosphere soil samples and small streams in natural and planted forest stands. Phylogenetic analyses of sequences from the nuclear ITS, LSU, β-tubulin and HSP90 loci and the mitochondrial cox1 and NADH1 genes revealed they belong to six new species of a new genus, officially described here as Nothophytophthora gen. nov., which clustered as sister group to Phytophthora. Nothophytophthora species share numerous morphological characters with Phytophthora: persistent (all Nothophytophthora spp.) and caducous (N. caduca, N. chlamydospora, N. valdiviana, N. vietnamensis) sporangia with variable shapes, internal differentiation of zoospores and internal, nested and extended (N. caduca, N. chlamydospora) and external (all Nothophytophthora spp.) sporangial proliferation; smooth-walled oogonia with amphigynous (N. amphigynosa) and paragynous (N. amphigynosa, N. intricata, N. vietnamensis) attachment of the antheridia; chlamydospores (N. chlamydospora) and hyphal swellings. Main differing features of the new genus are the presence of a conspicuous, opaque plug inside the sporangiophore close to the base of most mature sporangia in all known Nothophytophthora species and intraspecific co-occurrence of caducity and non-papillate sporangia with internal nested and extended proliferation in several Nothophytophthora species. Comparisons of morphological structures of both genera allow hypotheses about the morphology and ecology of their common ancestor which are discussed. Production of caducous sporangia by N. caduca, N. chlamydospora and N. valdiviana from Valdivian rainforests and N. vietnamensis from a mountain forest in Vietnam suggests a partially aerial lifestyle as adaptation to these humid habitats. Presence of tree dieback in all forests from which Nothophytophthora spp. were recovered and partial sporangial caducity of several Nothophytophthora species indicate a pathogenic rather than a saprophytic lifestyle. Isolation tests from symptomatic plant tissues in these forests and pathogenicity tests are urgently required to clarify the lifestyle of the six Nothophytophthora species.
- Widespread Phytophthora infestations in European nurseries put forest, semi-natural and horticultural ecosystems at high risk of Phytophthora diseasesPublication . Jung, Thomas; Orlikowski, L.; Henricot, B.; Abad-Campos, P.; Aday, A. G.; Aguin Casal, O.; Bakonyi, J.; Cacciola, S. O.; Cech, T.; Chavarriaga, D.; Corcobado, T.; Cravador, A.; Decourcelle, T.; Denton, G.; Diamandis, S.; Dogmus-Lehtijaervi, H. T.; Franceschini, A.; Ginetti, Beatrice; Green, S.; Glavendekic, M.; Hantula, J.; Hartmann, G.; Herrero, M.; Ivic, D.; Horta Jung, Marília; Lilja, A.; Keca, N.; Kramarets, V.; Lyubenova, A.; Machado, H.; Magnano di San Lio, G.; Mansilla Vazquez, P. J.; Marcais, B.; Matsiakh, I.; Milenkovic, I.; Moricca, S.; Nagy, Z. A.; Nechwatal, J.; Olsson, C.; Oszako, T.; Pane, A.; Paplomatas, E. J.; Pintos Varela, C.; Prospero, S.; Rial Martinez, C.; Rigling, D.; Robin, C.; Rytkoenen, A.; Sanchez, M. E.; Sanz Ros, A. V.; Scanu, B.; Schlenzig, A.; Schumacher, J.; Slavov, S.; Solla, A.; Sousa, E.; Stenlid, J.; Talgo, V.; Tomic, Z.; Tsopelas, P.; Vannini, A.; Vettraino, A. M.; Wenneker, M.; Woodward, S.; Perez-Sierra, A.An analysis of incidence of Phytophthora spp. in 732 European nurseries producing forest transplants, larger specimen trees, landscape plants and ornamentals, plus 2525 areas in which trees and shrubs were planted, is presented based on work conducted by 38 research groups in 23 European countries between 1972 and 2013. Forty-nine Phytophthora taxa were recorded in 670 nurseries (91.5%); within these nurseries, 1614 of 1992 nursery stands (81.0%) were infested, although most affected plants appeared healthy. In forest and landscape plantings, 56 Phytophthora taxa were recovered from 1667 of 2525 tested sites (66.0%). Affected plants frequently showed symptoms such as crown thinning, chlorosis and dieback caused by extensive fine root losses and/or collar rot. Many well-known highly damaging host-Phytophthora combinations were frequently detected but 297 and 407 new Phytophthora-host associations were also observed in nurseries and plantings, respectively. On average, 1.3 Phytophthora species/taxa per infested nursery stand and planting site were isolated. At least 47 of the 68 Phytophthora species/taxa detected in nurseries and plantings were exotic species several of which are considered well established in both nurseries and plantings in Europe. Seven known Phytophthora species/taxa were found for the first time in Europe, while 10 taxa had not been previously recorded from nurseries or plantings; in addition, 5 taxa were first detections on woody plant species. Seven Phytophthora taxa were previously unknown to science. The reasons for these failures of plant biosecurity in Europe, implications for forest and semi-natural ecosystems and possible ways to improve biosecurity are discussed.
- 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.