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

• Published in: Plant pathology Vol. 60; no. 5; pp. 929 - 937
• Main Authors: Thirugnanasambandam, A., Wright, K. M., Havis, N., Whisson, S. C., Newton, A. C.
• Format: Journal Article
• Language: English
• Published: Oxford, UK Blackwell Publishing Ltd 01.10.2011; Blackwell; Wiley Subscription Services, Inc
• Subjects: Agrobacterium; barley; Biological and medical sciences; Developmental stages; Endophytes; Fluorescent indicators; fluorescent protein; Fundamental and applied biological sciences. Psychology; Fungal plant pathogens; Green fluorescent protein; Hordeum vulgare; imaging; Infection; Inoculation; Leafspot; Leaves; Life cycle; Mycelia; Pathogens; Phytopathology. Animal pests. Plant and forest protection; Plasmids; Protoplasts; ramularia leaf spot; Transformation; Monocotyledones; Plant pathology; Fluorescent tracer; Plant pathogen; Cereal crop; Fungi; fluorescent protein; Agrobacterium; Transfection; Gramineae; Imaging; Angiospermae; Development; Bacteria; Fungi Imperfecti; C3-Type; Gram negative bacteria; Barley; ramularia leaf spot; Vegetals; Genetic transformation; Hordeum vulgare; Biochemical compound; Plant leaf; transformation; Protein; Infection; Vegetative apparatus; Cereal; Prokaryote; Spermatophyta; Rhizobiaceae; Fluorescent compound
• ISSN: 0032-0862; 1365-3059
• DOI: 10.1111/j.1365-3059.2011.02440.x

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.