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

摘要

To compare the infection process of 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 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 - gene driven by the glyceraldehyde 3P dehydrogenase promoter of . 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 transformants was monitored by immunodetection and epifluorescence and confocal microscopy. The Atd9- 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 was at least three times faster than on apples.