Gene-specific disruption in the filamentous fungus <Emphasis Type="Italic">Cercospora nicotianae</Emphasis> using a split-marker approach |
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Authors: | Bang-Jau You Miin-Huey Lee Kuang-Ren Chung |
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Institution: | (1) School of Chinese Medicine Resources, College of Pharmacy, China Medical University, 91, Hsueh-Shih Road, Taichung, 404, Taiwan;(2) Department of Plant Pathology, National Chung-Hsing University, 250 Kuo Kuang Road, Taichung, 402, Taiwan;(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 |
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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.
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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Keywords: | Gene replacement Filamentous fungi Pathogenicity Plant pathogen Recombination Split marker Toxins Virulence |
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