PEG-mediated and Agrobacterium-mediated transformation in the mycopathogen Verticillium fungicola

Verticillium fungicola, a severe mycopathogen of the cultivated mushroom Agaricus bisporus, was successfully transformed using both PEG-mediated and Agrobacterium-mediated techniques. PEG-mediated co-transformation was successful with hygromycin B resistance (hph), uidA (β-glucuronidase GUS), and gr...

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Bibliographic Details
Published in:Mycological research 2002-01, Vol.106 (1), p.4-11
Main Authors: AMEY, Richard C., ATHEY-POLLARD, Anna, BURNS, Claire, MILLS, Peter R., BAILEY, Andy, FOSTER, Gary D.
Format: Article
Language:English
Subjects:
Agaricus bisporus
Agrobacterium
Biological and medical sciences
Fundamental and applied biological sciences. Psychology
Fungal plant pathogens
Generalities. Techniques
GFP gene
hph gene
Phytopathology. Animal pests. Plant and forest protection
Polyethylene glycol
uidA gene
Verticillium fungicola
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Summary:Verticillium fungicola, a severe mycopathogen of the cultivated mushroom Agaricus bisporus, was successfully transformed using both PEG-mediated and Agrobacterium-mediated techniques. PEG-mediated co-transformation was successful with hygromycin B resistance (hph), uidA (β-glucuronidase GUS), and green fluorescent protein (GFP) genes. Agrobacterium-mediated transformation was successful with the hph gene. Transformation frequencies of up to 102 transformants per μg DNA and 4068 transformants per 105 conidia were obtained for PEG-mediated and Agrobacterium-mediated transformation respectively. Expression of integrated genes in co-transformants was stable after 18 months of successive sub-culturing on non-selective medium, and following storage at −80 °C in glycerol. Molecular analysis of PEG-mediated transformants showed integration of the transforming genes into the target genome. Molecular analysis of Agrobacterium-mediated transformants showed integration of transforming DNA as single copies within the target genome. Co-transformants exhibited symptoms of disease in inoculation experiments and were at least as virulent as the wild-type fungus. GFP and GUS expression were observed in-vivo with the GFP-tagged strain showing great potential as a tool in epidemiological and host-pathogen interaction studies. The development of transformation systems for V. fungicola will allow in-depth molecular studies of the interaction of this organism with A. bisporus.
ISSN:0953-7562
1469-8102
DOI:10.1017/S0953756201005251