Browsing by Author "Carvalho, Marisa"
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- Biochemical and molecular classification of yeasts from Trás-os-Montes honeyPublication . Carvalho, Marisa; Pinhel, Isabel; Rocha, Amélia; Choupina, Altino; Estevinho, Leticia M.Anexo
- 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, 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.
- Identification and characterization of molecular factors associated with the Phytophthora cinnamomi infection mechanismsPublication . Choupina, Altino; Meirinho, Sofia G.; Carvalho, Marisa; Jorge, Lurdes; Sousa, Maria João; Cravador, Alfredo
- Identification and characterization of molecular factors associated with the Phytophthora cinnamomi infection mechanismsPublication . Choupina, Altino; Meirinho, Sofia G.; Carvalho, Marisa; Jorge, Lurdes; Sousa, Maria João; Cravador, Alfredo
- Identification of a Phytophthora cinnamomi glucanase inhibitor protein: a molecular factor associated to infection mechanism.Publication . Carvalho, Marisa; Meirinho, Sofia G.; Jorge, Lurdes; Dominguez, Ángel; Choupina, AltinoThe oomycete P. cinnamomi, the causal agent of Castanea sativa ink disease, is one of the most distinctive species of Phytophthora genus, and has been associated with the decline of several forest, ornamental and fruit trees and shrubs, causing enormous economic losses worldwide. Phytophthora cell walls are composed of glucanase and have no chitin. Production of glucanase inhibitor proteins (G IPs) by Phytophthora species are thought to provide them a counter-defense against plant hosts P- l ,3-g11lcanascs (Rose el aI., 2002), that otherwise would degrade these pathogen cell walls. GIPs belongs to the chymotrypsin family of serine proteases but are catalytically nonfunctional because one or more residues of the essential catalytic triad are absent.
- Identification of a Phytophthora cinnamomi glucanase inhibitor protein: a molecular factor associated to infection mechanism.Publication . Carvalho, Marisa; Meirinho, Sofia G.; Jorge, Lurdes; Dominguez, Ángel; Choupina, AltinoThe oomycete P. cinnamomi, the causal agent of Castanea sativa ink disease, is one of the most destructive species of Phytophthora genus, and has been associated with the decline of several forest, ornamental and fruit trees and shrubs, causing enormous economic losses worldwide. Phytophthora cell walls are composed of glucans and have no chitin. Production of glucanase inhibitor proteins (GIPs) by Phytophthora species are thought to provide them a counter-defense against plant hosts β-1,3-glucanases (Rose et al., 2002), that otherwise would degrade these pathogen cell walls. GIPs belongs to the chymotrypsin family of serine proteases but are catalytically nonfunctional because one or more residues of the essential catalytic triad are absent. We report the identification of the gene encoding the first known P. cinnamomi GIP, presumably involved in the pathogen infection mechanism. Total genomic DNA was obtained from strain P. cinnamomi Pr120 and polymerase chain reaction was used to amplify a 308bp fragment of the GIP gene, using degenerate oligonucleotide primers, which were designed based on homology of previous published sequences of Phytophthora sp. GIP’s from EMBL databases. Full gene sequence length (1171bp) was obtained by flanking the known sequence with asymmetric PCR. P. cinnamomi GIP gene encodes a 269 amino acids protein with 28,818.2Da and a calculated global iso-electric point value of 8.54. It shares great identity and similarity with already described GIPs of P. sojae and P. infestans (E-values from 3.4e-49 to 2.6e-38), showing the importance of these proteins as effectors in plant-pathogen infection process. The UniProtKB/TrEMBL accession number for the sequence reported in this paper is B0B0H5_PHYCI.
- Identification of honey yeast species based on RELP analyses of the its regionPublication . Carvalho, Marisa; Rocha, Amélia; Estevinho, Leticia M.; Choupina, AltinoIn he present study, the restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene were used to identify a total of seven honey yeast species belonging to six different genera. Polymerase chain reaction (PCR) products of this rDNA region showed a high length variation for the different species. The size of the PCR products and the restriction patterns obtained with endonucleases HhaI, HaeIII and HinfI yielded a unique profile for each species, except for Zygoscharomyces mellis. The use of this molecular approach is proposed as a new rapid and easy method of routine honey yeast identification. En el presente estudio, el perfil de restriccin generado por la regin que abarca los separadores transcritos internos (ITS1 y ITS2) y el gen 5,8S rRNA se us para identificar un total de 7 levaduras de mieles pertenecientes a 6 gneros diferentes. Los productos de la reaccin en cadena de la polimerasa (PCR) en esta regin rDNA mostraron una alta variabilidad en la longitud para las diferentes especies. El tamao de los productos de PCR y el perfil de restriccin obtenido con endonucleasas HhaI, HaeIII y HinfI rindieron un perfil unico para cada especie, excepto para Zygoscharomyces mellis. El uso de esta aproximacion molecular se propone como un nuevo metodo rapido y facil de usar para la identificacion rutinaria de levaduras de mieles.
- Isolation and characterization by asymmetric PCR of the ENDO1G gene for Glucan endo-1,3-b-D-glucosidase in Phytophthora cinnamomi associated whit the ink disease of Castanea sativa Mill.Publication . Meirinho, Sofia G.; Carvalho, Marisa; Dominguez, Ángel; Choupina, AltinoInk disease is one of the most destructive diseases in Castanea sativa. The most common symptoms are root necrosies and a reduction in root growth, which invariably lead to the death of the trees. Phytophthora cinnamomi is an oomycete associated with this disease whose life cycle develops integrally in the soil. In the present work, was a fragment with 1231bp of the glucan endo-1,3-b-D-glucosidase gene obtained by amplification, using conserved primers and the full-length gene sequence by flanking this known sequence by asymmetric PCR. This fragment was obtained from genomic DNA of Phytophthora cinnamomi isolated in the European Regions of Castilla-Leon (Spain) and Trás-os-Montes (Portugal) and associated with the ink disease of Castanea sativa Mill.
- Yeast species associated with honey: different identification methodsPublication . Carvalho, Marisa; Estevinho, Leticia M.; Choupina, AltinoIn the present study, three different methods were used to identify yeast isolated from Trás-os-Montes, Portuguese honey. A total of 24 isolates were identified using a partial sequence of the 26S rRNA gene (rDNA), restriction patterns generated from the region spanning the internal transcribed spacers (ITS1 and ITS2) of the 5.8S rRNA gene and the API 20C AUX kit. Nine different yeast species were identified representing six different genera. Among the isolated honey samples, Rhodotorula mucilaginosa, Candida magnoliae and Zygosaccharomyces mellis were the predominant species. Partial sequence of the 26S rDNA yielded the best results in terms of correct identification, followed by-5.8S-ITS analysis. The commercial identification kit API 20C AUX was able to correctly identify only 58% of the isolates. Two new 5.8S-ITS profiles were described, corresponding to Trichosporon mucoides and Candida sorbosivorans.