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Gene-specific disruption in the filamentous fungus Cercospora nicotianae using a split-marker approach

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Abstract

To determine if DNA configuration, gene locus, and flanking sequences will affect homologous recombination in the phytopathogenic fungus Cercospora nicotianae, we evaluated and compared disruption efficiency targeting four cercosporin toxin biosynthetic genes encoding a polyketide synthase (CTB1), a monooxygenase/O-methyltransferase (CTB3), a NADPH-dependent oxidoreductase (CTB5), and a FAD/FMN-dependent oxidoreductase (CTB7). Transformation of C. nicotianae using a circular plasmid resulted in low disruption frequency. The use of endonucleases or a selectable marker DNA fragment flanked by homologous sequence either at one end or at both ends in the transformation procedures, increased disruption efficiency in some but not all CTB genes. A split-marker approach, using two DNA fragments overlapping within the selectable marker, increased the frequency of targeted gene disruption and homologous integration as high as 50%, depending on the target gene and on the length of homologous DNA sequence flanking the selectable marker. The results indicate that the split-marker approach favorably decreased ectopic integration and thus, greatly facilitated targeted gene disruption in this important fungal pathogen.

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Abbreviations

CSPD:

Disodium 3-[4-methoxyspiro{1,2-dioxetane-3,2′-(5′-chloro)tricyclo [3.3.1.1]decan}-4-yl]phenyl phosphate

BAR :

Acetyltransferase gene conferring phosphinothricin resistance

HYG :

Phosphotransferase B gene conferring hygromycin resistance

dUTP:

2′-Deoxyuracil 5′-triphosphate

DIG:

Digoxigenin

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Acknowledgment

The authors thank Dr. Daryl Henderson for the initial editing.

Author information

Authors and Affiliations

  1. School of Chinese Medicine Resources, College of Pharmacy, China Medical University, 91, Hsueh-Shih Road, Taichung, 404, Taiwan

    Bang-Jau You

  2. Department of Plant Pathology, National Chung-Hsing University, 250 Kuo Kuang Road, Taichung, 402, Taiwan

    Miin-Huey Lee

  3. Department of Plant Pathology, Citrus Research and Education Center, Institute of Food and Agricultural Sciences (IFAS), University of Florida, 700 Experiment Station Road, Lake Alfred, FL, 33850, USA

    Kuang-Ren Chung

Authors
  1. Bang-Jau You
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  2. Miin-Huey Lee
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  3. Kuang-Ren Chung
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Corresponding author

Correspondence to Kuang-Ren Chung.

Additional information

Communicated by Axel Brakhage.

The GenBank/EMBL/DDBJ accession numbers for the sequence data reported in this article are: CTB1, AY649543, CTB3, DQ355149, CTB5, DQ991507, and CTB7, DQ991509.

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You, BJ., Lee, MH. & Chung, KR. Gene-specific disruption in the filamentous fungus Cercospora nicotianae using a split-marker approach. Arch Microbiol 191, 615–622 (2009). https://doi.org/10.1007/s00203-009-0489-4

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  • DOI: https://doi.org/10.1007/s00203-009-0489-4

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