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Agrobacterium tumefaciens-mediated transformation of the violet root-rot fungus, Helicobasidium mompa, and the effect of activated carbon

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Mycoscience

Abstract

Agrobacterium tumefaciens-mediated transformation (ATMT) has been successfully applied to the violet root-rot fungus Helicobasidium mompa, which is the causal agent of violet root-rot disease. The A. tumefaciens strains carried a binary plasmid vector containing the hygromycin B phosphotransferase gene (hph) controlled by the heterologous fungal Agaricus bisporus P-gpd (glyceraldehyde-3-phosphate dehydrogenase) promoter and the trpC terminator. The transformation system was optimized using defined cocultivation conditions. When H. mompa strain V17 was cocultivated with A. tumefaciens strain AGL-1 using 5% agar, we obtained more hygromycin-resistant colonies than with strains EHA105 or MAFF301222 using 2% agar. In addition, our results suggest that the activated carbon is necessary in ATMT to reduce background growth of H. mompa. The presence of the hph gene in transformants was detected by polymerase chain reaction (PCR), and single-copy integration of the marker gene was demonstrated by Southern blot analysis. Thus, the ATMT system can be considered a promising tool for insertional mutagenesis studies of H. mompa.

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Acknowledgments

This research was supported by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry (BRAIN), Japan. The authors thank Dr. Hitoshi Nakamura (National Institute of Agro-Environmental Sciences, Japan) for providing the H. mompa strains and Dr. Yoshiyuki Niimi (Prefectural University of Hiroshima, Japan) for providing the Agrobacterium strain. We also thank Dr. Kiminori Shimizu (Chiba University) for providing technical advice.

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Authors and Affiliations

  1. Department of Life System Science, Graduate School of Comprehensive Scientific Research, Prefectural University of Hiroshima, 562 Nanatsuka, Shobara, Hiroshima, 727-0023, Japan

    Sanae Kano, Takuma Kurita & Tsutomu Morinaga

  2. National Institute of Fruit Tree Science, National Agricultural Research Organization (NARO), 92-24 Shimokuriyagawa, Morioka, Iwate, 020-0123, Japan

    Satoko Kanematsu

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  1. Sanae Kano
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  2. Takuma Kurita
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  3. Satoko Kanematsu
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  4. Tsutomu Morinaga
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Correspondence to Tsutomu Morinaga.

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Kano, S., Kurita, T., Kanematsu, S. et al. Agrobacterium tumefaciens-mediated transformation of the violet root-rot fungus, Helicobasidium mompa, and the effect of activated carbon. Mycoscience 52, 24–30 (2011). https://doi.org/10.1007/s10267-010-0067-2

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  • DOI: https://doi.org/10.1007/s10267-010-0067-2

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