Cloning, characterization, in vitro and in planta expression of a necrosis-inducing Phytophthora protein 1 gene npp1 from Phytophthora cinnamomi
Autor(a) principal: | |
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Data de Publicação: | 2019 |
Outros Autores: | , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
Texto Completo: | http://hdl.handle.net/1822/62528 |
Resumo: | The 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. |
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Cloning, characterization, in vitro and in planta expression of a necrosis-inducing Phytophthora protein 1 gene npp1 from Phytophthora cinnamomiCastanea sativaHE-TAIL PCRnpp1Phytophthora cinnamomiPichia pastorisRT-qPCREngenharia e Tecnologia::Biotecnologia AmbientalScience & TechnologyThe 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.Project COMBATINTA/SP2.P11/02 Interreg IIIA—Cross-Border Cooperation Spain-Portugal, financed by The European Regional Development Fund and by national funding from the Portuguese Ministério da Ciência e do Ensino Superior (MCES) (PTDC/AGR-AAM/67628/2006).info:eu-repo/semantics/publishedVersionSpringer NatureUniversidade do MinhoMartins, Ivone M.Meirinho, SofiaCosta, RodrigoCravador, AlfredoChoupina, Altino2019-122019-12-01T00:00:00Zinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articleapplication/pdfhttp://hdl.handle.net/1822/62528engMartins, Ivone M.; Meirinho, Sofia; Costa, Rodrigo; Cravador, Alfredo; Choupina, Altino, Cloning, characterization, in vitro and in planta expression of a necrosis-inducing Phytophthora protein 1 gene npp1 from Phytophthora cinnamomi. Molecular Biology Reports, 46(6), 6453-6462, 20190301-48511573-497810.1007/s11033-019-05091-031571106http://www.springer.com/journal/11033/aboutinfo:eu-repo/semantics/openAccessreponame:Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)instname:Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãoinstacron:RCAAP2023-07-21T12:21:28Zoai:repositorium.sdum.uminho.pt:1822/62528Portal AgregadorONGhttps://www.rcaap.pt/oai/openaireopendoar:71602024-03-19T19:14:47.085086Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informaçãofalse |
dc.title.none.fl_str_mv |
Cloning, characterization, in vitro and in planta expression of a necrosis-inducing Phytophthora protein 1 gene npp1 from Phytophthora cinnamomi |
title |
Cloning, characterization, in vitro and in planta expression of a necrosis-inducing Phytophthora protein 1 gene npp1 from Phytophthora cinnamomi |
spellingShingle |
Cloning, characterization, in vitro and in planta expression of a necrosis-inducing Phytophthora protein 1 gene npp1 from Phytophthora cinnamomi Martins, Ivone M. Castanea sativa HE-TAIL PCR npp1 Phytophthora cinnamomi Pichia pastoris RT-qPCR Engenharia e Tecnologia::Biotecnologia Ambiental Science & Technology |
title_short |
Cloning, characterization, in vitro and in planta expression of a necrosis-inducing Phytophthora protein 1 gene npp1 from Phytophthora cinnamomi |
title_full |
Cloning, characterization, in vitro and in planta expression of a necrosis-inducing Phytophthora protein 1 gene npp1 from Phytophthora cinnamomi |
title_fullStr |
Cloning, characterization, in vitro and in planta expression of a necrosis-inducing Phytophthora protein 1 gene npp1 from Phytophthora cinnamomi |
title_full_unstemmed |
Cloning, characterization, in vitro and in planta expression of a necrosis-inducing Phytophthora protein 1 gene npp1 from Phytophthora cinnamomi |
title_sort |
Cloning, characterization, in vitro and in planta expression of a necrosis-inducing Phytophthora protein 1 gene npp1 from Phytophthora cinnamomi |
author |
Martins, Ivone M. |
author_facet |
Martins, Ivone M. Meirinho, Sofia Costa, Rodrigo Cravador, Alfredo Choupina, Altino |
author_role |
author |
author2 |
Meirinho, Sofia Costa, Rodrigo Cravador, Alfredo Choupina, Altino |
author2_role |
author author author author |
dc.contributor.none.fl_str_mv |
Universidade do Minho |
dc.contributor.author.fl_str_mv |
Martins, Ivone M. Meirinho, Sofia Costa, Rodrigo Cravador, Alfredo Choupina, Altino |
dc.subject.por.fl_str_mv |
Castanea sativa HE-TAIL PCR npp1 Phytophthora cinnamomi Pichia pastoris RT-qPCR Engenharia e Tecnologia::Biotecnologia Ambiental Science & Technology |
topic |
Castanea sativa HE-TAIL PCR npp1 Phytophthora cinnamomi Pichia pastoris RT-qPCR Engenharia e Tecnologia::Biotecnologia Ambiental Science & Technology |
description |
The 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. |
publishDate |
2019 |
dc.date.none.fl_str_mv |
2019-12 2019-12-01T00:00:00Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://hdl.handle.net/1822/62528 |
url |
http://hdl.handle.net/1822/62528 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Martins, Ivone M.; Meirinho, Sofia; Costa, Rodrigo; Cravador, Alfredo; Choupina, Altino, Cloning, characterization, in vitro and in planta expression of a necrosis-inducing Phytophthora protein 1 gene npp1 from Phytophthora cinnamomi. Molecular Biology Reports, 46(6), 6453-6462, 2019 0301-4851 1573-4978 10.1007/s11033-019-05091-0 31571106 http://www.springer.com/journal/11033/about |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Springer Nature |
publisher.none.fl_str_mv |
Springer Nature |
dc.source.none.fl_str_mv |
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Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) |
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Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos) - Agência para a Sociedade do Conhecimento (UMIC) - FCT - Sociedade da Informação |
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