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Construction of a system for exploring mitotic homologous recombination in the genome of Pyricularia oryzae

  • Fungal Diseases
  • Published:
Journal of General Plant Pathology Aims and scope Submit manuscript

Abstract

To investigate mitotic homologous recombination (HR) in Pyricularia oryzae, we created an HR detection system. The system consists of two non-functional enhanced yellow fluorescent protein (YFP) and blasticidin S deaminase (BSD) fusion genes (YFP::BSD). If mitotic HR occurs between the two non-functional genes in the genome, restoration of the functional YFP::BSD gene can be expected. The expression of the functional YFP::BSD gene can be detected by both YFP fluorescence and resistance against BS. When the P. oryzae genome was transformed simultaneously with two non-functional genes, all six lines of transformants with both genes had some portion of their hyphae exhibiting YFP fluorescence and BS resistance during growth. Up to ca. 10 % of conidia harvested from the mycelium of each of the six lines had YFP fluorescence, suggesting that HR consistently occurs during mycelium growth. To determine whether and how the HR-mediated phenotypic changes occurred at the DNA level, we analyzed the genomic DNA of BS-resistant mycelia by PCR-RFLP and sequencing and were able to confirm the existence of a restored functional YFP::BSD gene and a non-functional recombinant gene. Collectively, these findings demonstrate that we established a successful HR detection system for P. oryzae, which can be used for other plant pathogens, and that mitotic HR actually occurs in P. oryzae and constitute the first experimental evidence for mitotic HR in a fungus.

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Acknowledgments

The authors thank Dr. Takehiko Shibata at the RIKEN Advanced Science Institute for valuable suggestions. This work was supported in part by Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for JSPS Fellows grant number 24.11224 and JSPS Grant-in-Aid for Scientific Research (C) grant number 24580070.

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Authors and Affiliations

  1. Graduate School of Agriculture, Meiji University, 1-1-1 Higashi-Mita, Tama-ku, Kawasaki, Kanagawa, 214-8571, Japan

    Takayuki Arazoe, Shuichi Ohsato, Katsuyoshi Yoneyama & Shigeru Kuwata

  2. Graduate School of Agriculture, Tokyo University of Agriculture and Technology, Tokyo, Japan

    Tsutomu Arie

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  1. Takayuki Arazoe
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  2. Shuichi Ohsato
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  3. Tsutomu Arie
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  4. Katsuyoshi Yoneyama
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  5. Shigeru Kuwata
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Correspondence to Shigeru Kuwata.

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Arazoe, T., Ohsato, S., Arie, T. et al. Construction of a system for exploring mitotic homologous recombination in the genome of Pyricularia oryzae . J Gen Plant Pathol 79, 422–430 (2013). https://doi.org/10.1007/s10327-013-0474-2

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  • DOI: https://doi.org/10.1007/s10327-013-0474-2

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