Browsing by Author "Cravador, Alfredo"
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- 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.
- 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.
- 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