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Updated: Jan 1, 2026
Author Spotlight: Investigating Fungal Pathogenicity Mechanisms in Maize
Published on: September 15, 2023
Labeling of Monilinia fructicola with GFP and Its Validation for Studies on Host-Pathogen Interactions in Stone and
Silvia Rodríguez-Pires1, Eduardo Antonio Espeso2, Nuria Baró-Montel3
1Department of Plant Protection, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria, Ctra. de La Coruña Km. 7, 28040 Madrid, Spain.
Abstract:
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.
Insights
Monilinia fructicola infects nectarines three times faster than apples. Researchers developed a fluorescently tagged strain of the pathogen to visualize and compare infection processes in vivo using confocal microscopy.
Area of Science:
- Plant Pathology
- Molecular Biology
- Microscopy
Background:
- Monilinia fructicola is a significant pathogen affecting stone fruits and pome fruits.
- Understanding the in vivo infection dynamics is crucial for developing effective disease management strategies.
Purpose of the Study:
- To compare the in vivo infection process of Monilinia fructicola on nectarines and apples.
- To develop a fluorescently tagged strain of M. fructicola for live imaging of infection.
Main Methods:
- Transformation of M. fructicola with Agrobacterium tumefaciens carrying the pPK2-hphgfp plasmid for Hph-GFP expression.
- Selection of transformants using hygromycin B resistance.
- Confirmation of T-DNA integration via PCR and analysis of insertion sites.
- Monitoring Hph-GFP expression using immunodetection and confocal microscopy.
Main Results:
- Successfully generated hygromycin-resistant M. fructicola transformants expressing Hph-GFP.
- Confirmed transgene integration at unique genomic locations with structural rearrangements.
- The Atd9 transformant exhibited normal growth and pathogenicity.
- Confocal microscopy revealed that M. fructicola infection is at least three times faster on nectarines compared to apples.
Conclusions:
- The developed fluorescently tagged M. fructicola strain is a valuable tool for studying host-pathogen interactions.
- Nectarines are significantly more susceptible to M. fructicola infection than apples, with a faster infection progression.
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