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

By Richard C. Amey, Anna Athey-Pollard, C. (Claire) Burns, P. R. Mills, Andy M. Bailey and Gary D. Foster

Abstract

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

Topics: SB
Publisher: Cambridge University Press
Year: 2002
OAI identifier: oai:wrap.warwick.ac.uk:789

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  1. (1998). Agrobacterium tumefaciens-mediated transformation of ®lamentous fungi. doi
  2. (2001). Agrobacterium tumefaciens-mediated transformation of Fusarium circinatum. doi
  3. (2001). Agrobacterium-mediated transformation of Fusarium oxysporum: an efficient tool for insertional mutagenesis and gene transfer. doi
  4. (1984). Binary Agrobacterium vectors for plant transformation. doi
  5. (1999). Co-transformation of the sclerotialmycoparasiteConiothyrium minitanswiththe hygromycin B resistance and b-glucuronidase markers. doi
  6. (1997). Enzymic activity of the mycoparasite Verticillium fungicola on Agaricus bisporus fruit body cell walls. doi
  7. (2000). Fraza 4 o, doi
  8. (1992). Gene transformation in plant pathogenic fungi.
  9. (1993). Host range of a plant pathogenic fungus is determined by a saponin detoxifying enzyme. doi
  10. (1993). Identi®cation and characterisation of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea. Plant Cell 5: 1575±1590. Corresponding Editor: doi
  11. (2000). Interaction between the mycoparasite Verticillium fungicola and the vegetative mycelial phase of Agaricus bisporus. doi
  12. (1994). Interfungal Parasitic Relationships. doi
  13. (1996). Intracellular infections of the cultivated mushroom Agaricus bisporus by the mycoparasite Verticillium fungicola var. fungicola. doi
  14. (1998). Isolation and heterologous expression of a gene encoding 4-hydroxyphenylpyruvate dioxygenasefromthe wheatleaf-spot pathogen,Mycosphaerella graminicola. doi
  15. (1986). Mushrooms: pest and disease control. Intercept, doi
  16. R.(2000)CotransformationofTrichoderma harzianum with b-glucuronidase and green ¯uorescent protein genes provides a useful tool for monitoring fungal growth and activity in natural soils. doi
  17. (1979). Regulation of the transfer of Ti-plasmids of Agrobacterium tumefaciens. doi
  18. (1995). The race-speci®c elicitor, NIP1, from the barley pathogen, Rhynchosporium secalis, determines avirulence on host plants of the RRS1 resistance genotype.
  19. (1995). Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae.
  20. (1999). Transformation of Aspergillus awamori by Agrobacterium tumefaciens-mediated homologous recombination. doi
  21. (1987). Transformation of Aspergillus based on the hygromycin resistance marker from Escherichia coli. doi
  22. (1993). Transformation of Bipolaris sorokiniana with the GUS gene and use for studying fungal colonisation of barley roots. doi
  23. (1994). Use of ELISA and GUStransformed strains to study competition between pathogenic and non-pathogenic Fusarium oxysporum for root colonisation. doi
  24. (1993). Use of fungal transformants expressing b-glucuronidase activity to detect infection and measure hyphal biomass inR. C. Amey and others 11 infected plant tissues. doi

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