Labeling of Monilinia fructicola with GFP and Its Validation for Studies on Host-Pathogen Interactions in Stone and Pome Fruit

To compare in vivo the infection process of Monilinia fructicola on nectarines and apples using confocal microscopy it is necessary to transform a pathogenic strain with a construct expressing a fluorescent chromophore such as GFP. Thus, germinated conidia of the pathogen were transformed with Agrob...

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Autores: Rodríguez-Pires, Silvia, Espeso, Eduardo Antonio, Baró-Montel, Nuria, Torres, Rosario, Melgarejo, Paloma, De Cal, Antonieta
Tipo de recurso: artículo
Fecha de publicación:2019
País:España
Institución:Institut de Recerca i Tecnologia Agroalimentàries (IRTA)
Repositorio:IRTA Pubpro. Open Digital Archive
OAI Identifier:oai:repositori.irta.cat:20.500.12327/605
Acceso en línea:http://hdl.handle.net/20.500.12327/605
https://doi.org/10.3390/genes10121033
Access Level:acceso abierto
Palabra clave:633
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spelling Labeling of Monilinia fructicola with GFP and Its Validation for Studies on Host-Pathogen Interactions in Stone and Pome FruitRodríguez-Pires, SilviaEspeso, Eduardo AntonioBaró-Montel, NuriaTorres, RosarioMelgarejo, PalomaDe Cal, Antonieta633To compare in vivo the infection process of Monilinia fructicola on nectarines and apples using confocal microscopy it is necessary to transform a pathogenic strain with a construct expressing a fluorescent chromophore such as GFP. Thus, germinated conidia of the pathogen were transformed with Agrobacterium tumefaciens carrying the plasmid pPK2-hphgfp that allowed the expression of a fluorescent Hph-GFP chimera. The transformants were selected according to their resistance to hygromycin B, provided by the constitutive expression of the hph-gfp gene driven by the glyceraldehyde 3P dehydrogenase promoter of Aspergillus nidulans. The presence of T-DNA construct in the genomic DNA was confirmed by PCR using a range of specific primers. Subsequent PCR-mediated analyses proved integration of the transgene at a different genomic location in each transformant and the existence of structural reorganizations at these insertion points. The expression of Hph-GFP in three independent M. fructicola transformants was monitored by immunodetection and epifluorescence and confocal microscopy. The Atd9-M. fructicola transformant displayed no morphological defects and showed growth and pathogenic characteristics similar to the wild type. Microscopy analysis of the Atd9 transformant evidenced that nectarine infection by M. fructicola was at least three times faster than on applesinfo:eu-repo/semantics/publishedVersionMDPIProducció VegetalPostcollita201920192019info:eu-repo/semantics/article14application/pdfhttp://hdl.handle.net/20.500.12327/605https://doi.org/10.3390/genes10121033reponame:IRTA Pubpro. Open Digital Archiveinstname:Institut de Recerca i Tecnologia Agroalimentàries (IRTA)InglésGenesMINECO/Programa Estatal de I+D+I orientada a los retos de la sociedad/AGL2014-55287-C2-2-R/ES/FACTORES DE PATOGENESIS Y BASE GENETICA DE LA RESISTENCIA DEL MELOCOTON PARA EL CONTROL SOSTENIBLE DE LA PODREDUMBRE PARDA/MINECO/Programa Estatal de I+D+I orientada a los retos de la sociedad/BFU2015-66806-R/ES/LA TOLERANCIA A CATIONES Y EL PH ALCALINO COMO DETERMINANTE DE VIRULENCIA FUNGICA Y BASE PARA LA BUSQUEDA DE DIANAS DE ANTIFUNGICOS/MICIU/Programa Estatal de I+D+I orientada a los retos de la sociedad/RTI2018-094263-B-I00/ES/INTERCONEXIONES ENTRE LA HOMEOSTASIS DE CATIONES Y EL TRAFICO INTRACELULAR EN ASPERGILLUS NIDULANS/MINECO/Programa Estatal de I+D+I orientada a los retos de la sociedad/AGL2017-84389-C2-1-R/ES/PODREDUMBRE PARDA CAUSADA POR MONILINIA SPP. EN FRUTA DE HUESO: INTERACCION HUESPED-PATOGENO-AMBIENTE/FEDER/ / /EU/ /Attribution 4.0 Internationalhttp://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccessoai:repositori.irta.cat:20.500.12327/6052026-06-16T08:51:17Z
dc.title.none.fl_str_mv Labeling of Monilinia fructicola with GFP and Its Validation for Studies on Host-Pathogen Interactions in Stone and Pome Fruit
title Labeling of Monilinia fructicola with GFP and Its Validation for Studies on Host-Pathogen Interactions in Stone and Pome Fruit
spellingShingle Labeling of Monilinia fructicola with GFP and Its Validation for Studies on Host-Pathogen Interactions in Stone and Pome Fruit
Rodríguez-Pires, Silvia
633
title_short Labeling of Monilinia fructicola with GFP and Its Validation for Studies on Host-Pathogen Interactions in Stone and Pome Fruit
title_full Labeling of Monilinia fructicola with GFP and Its Validation for Studies on Host-Pathogen Interactions in Stone and Pome Fruit
title_fullStr Labeling of Monilinia fructicola with GFP and Its Validation for Studies on Host-Pathogen Interactions in Stone and Pome Fruit
title_full_unstemmed Labeling of Monilinia fructicola with GFP and Its Validation for Studies on Host-Pathogen Interactions in Stone and Pome Fruit
title_sort Labeling of Monilinia fructicola with GFP and Its Validation for Studies on Host-Pathogen Interactions in Stone and Pome Fruit
dc.creator.none.fl_str_mv Rodríguez-Pires, Silvia
Espeso, Eduardo Antonio
Baró-Montel, Nuria
Torres, Rosario
Melgarejo, Paloma
De Cal, Antonieta
author Rodríguez-Pires, Silvia
author_facet Rodríguez-Pires, Silvia
Espeso, Eduardo Antonio
Baró-Montel, Nuria
Torres, Rosario
Melgarejo, Paloma
De Cal, Antonieta
author_role author
author2 Espeso, Eduardo Antonio
Baró-Montel, Nuria
Torres, Rosario
Melgarejo, Paloma
De Cal, Antonieta
author2_role author
author
author
author
author
dc.contributor.none.fl_str_mv Producció Vegetal
Postcollita
dc.subject.none.fl_str_mv 633
topic 633
description To compare in vivo the infection process of Monilinia fructicola on nectarines and apples using confocal microscopy it is necessary to transform a pathogenic strain with a construct expressing a fluorescent chromophore such as GFP. Thus, germinated conidia of the pathogen were transformed with Agrobacterium tumefaciens carrying the plasmid pPK2-hphgfp that allowed the expression of a fluorescent Hph-GFP chimera. The transformants were selected according to their resistance to hygromycin B, provided by the constitutive expression of the hph-gfp gene driven by the glyceraldehyde 3P dehydrogenase promoter of Aspergillus nidulans. The presence of T-DNA construct in the genomic DNA was confirmed by PCR using a range of specific primers. Subsequent PCR-mediated analyses proved integration of the transgene at a different genomic location in each transformant and the existence of structural reorganizations at these insertion points. The expression of Hph-GFP in three independent M. fructicola transformants was monitored by immunodetection and epifluorescence and confocal microscopy. The Atd9-M. fructicola transformant displayed no morphological defects and showed growth and pathogenic characteristics similar to the wild type. Microscopy analysis of the Atd9 transformant evidenced that nectarine infection by M. fructicola was at least three times faster than on apples
publishDate 2019
dc.date.none.fl_str_mv 2019
2019
2019
dc.type.none.fl_str_mv info:eu-repo/semantics/article
format article
dc.identifier.none.fl_str_mv http://hdl.handle.net/20.500.12327/605
https://doi.org/10.3390/genes10121033
url http://hdl.handle.net/20.500.12327/605
https://doi.org/10.3390/genes10121033
dc.language.none.fl_str_mv Inglés
language_invalid_str_mv Inglés
dc.relation.none.fl_str_mv Genes
MINECO/Programa Estatal de I+D+I orientada a los retos de la sociedad/AGL2014-55287-C2-2-R/ES/FACTORES DE PATOGENESIS Y BASE GENETICA DE LA RESISTENCIA DEL MELOCOTON PARA EL CONTROL SOSTENIBLE DE LA PODREDUMBRE PARDA/
MINECO/Programa Estatal de I+D+I orientada a los retos de la sociedad/BFU2015-66806-R/ES/LA TOLERANCIA A CATIONES Y EL PH ALCALINO COMO DETERMINANTE DE VIRULENCIA FUNGICA Y BASE PARA LA BUSQUEDA DE DIANAS DE ANTIFUNGICOS/
MICIU/Programa Estatal de I+D+I orientada a los retos de la sociedad/RTI2018-094263-B-I00/ES/INTERCONEXIONES ENTRE LA HOMEOSTASIS DE CATIONES Y EL TRAFICO INTRACELULAR EN ASPERGILLUS NIDULANS/
MINECO/Programa Estatal de I+D+I orientada a los retos de la sociedad/AGL2017-84389-C2-1-R/ES/PODREDUMBRE PARDA CAUSADA POR MONILINIA SPP. EN FRUTA DE HUESO: INTERACCION HUESPED-PATOGENO-AMBIENTE/
FEDER/ / /EU/ /
dc.rights.none.fl_str_mv Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
info:eu-repo/semantics/openAccess
rights_invalid_str_mv Attribution 4.0 International
http://creativecommons.org/licenses/by/4.0/
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv 14
application/pdf
dc.publisher.none.fl_str_mv MDPI
publisher.none.fl_str_mv MDPI
dc.source.none.fl_str_mv reponame:IRTA Pubpro. Open Digital Archive
instname:Institut de Recerca i Tecnologia Agroalimentàries (IRTA)
instname_str Institut de Recerca i Tecnologia Agroalimentàries (IRTA)
reponame_str IRTA Pubpro. Open Digital Archive
collection IRTA Pubpro. Open Digital Archive
repository.name.fl_str_mv
repository.mail.fl_str_mv
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score 15.301603

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