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Deletion of the cpku80 gene in the chestnut blight fungus, Cryphonectria parasitica, enhances gene disruption efficiency

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Abstract

The chestnut blight fungus, Cryphonectria parasitica, and associated virulence-attenuating hypoviruses have emerged as an important model system for studying molecular mechanisms underlying fungal–plant pathogenic interactions. As more gene sequence information becomes available as a result of C. parasitica express sequence tags (ESTs) and ongoing whole genome sequencing projects, the development of an efficient gene disruption system has become an urgent need for functional genomics studies of this important forestry pathogen. Here, we report the cloning of the C. parasitica gene cpku80 that encodes a key component of the nonhomologous end joining DNA repair pathway and the construction of a corresponding deletion mutant strain. The cpku80 mutant was indistinguishable from the parental wild-type strain EP155 in colony morphology, ability to support hypovirus replication, conidiation and virulence. As predicted, the Δcpku80 strain did exhibit an increased sensitivity to the mutagen methyl methanesulfonate. A test with three selected genes resulted in a gene disruption efficiency of about 80% for the Δcpku80 strain, a significant increase over the 2–5% levels of homologous recombination generally observed for the wild-type strain EP155. This efficient homologous recombination system provides a powerful tool for large-scale analysis of gene functions in C. parasitica.

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Acknowledgments

This work was supported in part by grants from the National Natural Science Foundation of China (30130020 and 39925003), National Hightech Program of China (2001AA223111 and 2004AA223100), and Guangxi Natural Science Foundation grant 0229001 to B.C., the Open Fund of Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering JS501 to X.L., the Guangxi Graduate Education Creativity Program grant 2007105930710M32 to Z.Y. and by Public Health Service grant GM55981 to D.L.N. Reports of improved gene disruption by disruption of the NHEJ pathways for two other plant pathogenic fungi (Claviceps purpurea and Magnaporthe grisea) appeared on line while this manuscript was under review.

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Authors and Affiliations

  1. Guangxi Key Laboratory of Subtropical Bioresource Conservation and Utilization, The Key Laboratory of Ministry of Education of China for Microbial and Plant Genetic Engineering, and College of Life Science and Technology, Guangxi University, 100 Daxue Road, Nanning, Guangxi, 530004, People’s Republic of China

    Xiuwan Lan, Ziting Yao, Yan Zhou, Jinjie Shang, Haiyan Lin & Baoshan Chen

  2. Center for Biosystems Research, University of Maryland Biotechnology Institute, Shady Grove Campus, 9600 Gudelsky Drive, Rockville, MD, 20850, USA

    Donald L. Nuss

  3. Department of Biochemistry and Molecular Biology, Guangxi Medical University, 22 Shuangyong Road, Nanning, Guangxi, 530021, People’s Republic of China

    Xiuwan Lan

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  1. Xiuwan Lan
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  2. Ziting Yao
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  3. Yan Zhou
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  4. Jinjie Shang
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  5. Haiyan Lin
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  6. Donald L. Nuss
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  7. Baoshan Chen
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Corresponding author

Correspondence to Baoshan Chen.

Additional information

Communicated by J. Heitman.

The authors Xiuwan Lan and Ziting Yao contributed equally to this work.

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Lan, X., Yao, Z., Zhou, Y. et al. Deletion of the cpku80 gene in the chestnut blight fungus, Cryphonectria parasitica, enhances gene disruption efficiency. Curr Genet 53, 59–66 (2008). https://doi.org/10.1007/s00294-007-0162-x

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