Agrobacterium-mediated transformation of the barley pathogen Ramularia collo-cygni with fluorescent marker tags and live tissue imaging of infection development

Ramularia collo-cygni, the causal agent of ramularia leaf spot disease in barley was transformed to express the green fluorescent protein and DsRed fluorescent protein using a combined protoplast and Agrobacterium tumefaciens-mediated transformation technique. Regenerated protoplasts from mycelial fragments were transformed with A. tumefaciens strain AGL1 containing either pCAMBgfp or pCAMBDsRed plasmids. Two different isolates of R. collo-cygni were transformed and several stable transformants were recovered. These transformants infected detached barley leaves and characteristic swan's-neck-shaped conidiophores were seen on detached leaves 9 days after inoculation, as well as spore-like structures formed from hyphal swellings. This first successful transformation of R. collo-cygni has enabled better description of the stages of disease development and pathogen behaviour during the endophytic/asymptomatic stage of its life cycle.