Using the Green Fluorescent Protein to determine virulence and gene expression of Erwinia amylovora and other markers for strain differentiation

Several markers were used to distinguish individual Erwinia amylovora strains during colonization of plants. E. amylovora strains were labelled with the Green Fluorescent Protein (GFP) and visualized in plant tissue by fluorescence microscopy. Inoculation of the intercostal region of apple leaves caused slow movement of the bacteria in the apoplast followed by invasion into the vascular system. After inoculation at tips of apple leaves migration of the bacteria was observed through the xylem vessels and outbreaks into the adjacent intercellular space of the parenchyma. Non-pathogenic mutants did not move from the inoculation site. Plant resistance enhancers caused a decrease in the migration rate. The migration rate in the central vein of apple leaves corresponded to disease ratings from symptom formation on shoots of various apple cultivars. GFP was also used as a reporter to assay promoter activity in the srl- and scr-operons. Fluorescence was analyzed by flow cytometry (FACS). The promoter fusions were induced by the respective carbohydrate, and were repressed in the presence of glucose. Other markers for strain differentiation were the size of short sequence DNA repeats and labelling for streptomycin-resistance. Accordingly, the marked strains were compared for their plant tissue colonization.