Development of a green fluorescent protein (GFP) expression vector for transformation of potato ring rot bacterium

Potato bacterial ring rot caused by Clavibacter michiganensis subsp. sepedonicus (CMS) (formerly Corynebacterium sepedonicus), a gram-positive bacterium, is a big concern to seed and processing potato producers in Canada and worldwide. A plasmid, pHN216 (13.8 kbp), previously modified from two wild type plasmids isolated from C. michiganensis subsp. michiganensis (CMM) was used as a cloning vector. Two variants of jellyfish (Aquaria victoria ) green fluorescent protein (GFP) gene (gfp) were inserted in plasmid pHN216 in order to express GFP in CMS. In this study experiments were conducted to transform both CMS and CMM with the recombinant plasmid. Although CMS was the target of primary interest, transformation of CMM was also done. CMM is a pathogen of tomato and does not infect potato. In this study, two recombinant plasmids, pHNG and pHNCG were constructed. Escherichia coli, CMS and CMM cells were then transformed with these plasmids. A new method for the preparation of competent cells and for electroporation was developed in this study for enhancing the efficiency of transformation and all three bacterial species were successfully transformed with either pHNG or pHNCG. (Abstract shortened by UMI.).