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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 principales: Rodríguez-Pires, Silvia, Espeso, Eduardo Antonio, Baró-Montel, Nuria, Torres, Rosario, Melgarejo, Paloma, De Cal, Antonieta
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6947648/
https://www.ncbi.nlm.nih.gov/pubmed/31835779
http://dx.doi.org/10.3390/genes10121033
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author Rodríguez-Pires, Silvia
Espeso, Eduardo Antonio
Baró-Montel, Nuria
Torres, Rosario
Melgarejo, Paloma
De Cal, Antonieta
author_facet Rodríguez-Pires, Silvia
Espeso, Eduardo Antonio
Baró-Montel, Nuria
Torres, Rosario
Melgarejo, Paloma
De Cal, Antonieta
author_sort Rodríguez-Pires, Silvia
collection PubMed
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.
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spelling pubmed-69476482020-01-13 Labeling of Monilinia fructicola with GFP and Its Validation for Studies on Host-Pathogen Interactions in Stone and Pome Fruit Rodríguez-Pires, Silvia Espeso, Eduardo Antonio Baró-Montel, Nuria Torres, Rosario Melgarejo, Paloma De Cal, Antonieta Genes (Basel) Article 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. MDPI 2019-12-11 /pmc/articles/PMC6947648/ /pubmed/31835779 http://dx.doi.org/10.3390/genes10121033 Text en © 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Rodríguez-Pires, Silvia
Espeso, Eduardo Antonio
Baró-Montel, Nuria
Torres, Rosario
Melgarejo, Paloma
De Cal, Antonieta
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
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_short 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
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6947648/
https://www.ncbi.nlm.nih.gov/pubmed/31835779
http://dx.doi.org/10.3390/genes10121033
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