Migration and gene regulation of Erwinia amylovora in host plants assayed with the green fluorescent protein

In order to visualize the migration of Erwinia amylovora in plant tissue, the pathogen was labeled with the gene of coding the Green Fluorescent Protein (GFP) from the jellyfish Aequorea victoria. After inoculation of apple leaves with scissors fast migration of the bacteria through the xylem vessels and subsequently their penetration into the adjacent intercellular space of the parenchyma was observed. Inoculation of the intercostal region of apple leaves caused slow movement of the bacteria in the apoplast followed by the invasion of the vascular system. Labeled bacteria could also be seen in association with anchor sites of leaf hairs. The bacteria could enter the xylem after penetration of intact plant tissue. The virulence of E, amylovora strains was examined, by labeling the strains with gfp and measuring their migration rate in the middle vein of apple leaves. In addition to the sorbitol operon, we have determined the nucleotide sequence of the sucrose operon. By fusing the gfp-gene to the srl- and scr-promoter from E. amylovora with expression on a intermediate copy number plasmid, the regulation of these operons could be studied in culture and in plant tissue by flow cytometry. Gene induction was measured in plant tissue with E. amylovora containing the sorbitol-promoter: :gfp fusion. Both fusions showed catabolite repression for growth of the cells in the presence of glucose and induction by sorbitol or sucrose, respectively.