Abstract
Targeted gene replacement is one of the primary strategies for functional characterization of fungal genes and several methods have been developed for this purpose over the years. The increased availability of genome sequence information in the present times has enabled wider adoption of protocols based on the knowledge of the gene sequence and its surrounding region. Among such targeted gene replacement approaches, the spilt-marker method has gained popularity in filamentous fungi. This method involves only two rounds of PCR and does not require any subcloning. It is based on the availability of a marker gene (e.g., the hygromycin gene) and sequences of the gene of interest, as well as around 1 kb long regions flanking the gene on either side. The technique includes PCR amplification of the flanking regions of the gene of interest and the marker gene followed by a fusion PCR which leads to the creation of two molecular cassettes, each containing a part of the marker gene fused to one flanking region. These molecular cassettes are then simultaneously used for transformation of protoplasts. Three homologous recombination events, one within each flanking region and one in the marker gene, lead to the replacement of the gene of interest with a functional marker gene. The transformants are then grown on selective media and emerging colonies can be screened for presence of the marker and absence of the gene being replaced using various methods.
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Acknowledgments
I would like to thank Dr. H.C. Kistler in whose laboratory the protocol mentioned here was initially standardized for F. graminearum and members of his lab, particularly Karen Hilburn for her input in protocol development and manuscript preparation. Thanks are also due to Dr. Jin-Rong Xu for assisting with standardization of the method and providing additional information for the chapter.
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Goswami, R.S. (2012). Targeted Gene Replacement in Fungi Using a Split-Marker Approach. In: Bolton, M., Thomma, B. (eds) Plant Fungal Pathogens. Methods in Molecular Biology, vol 835. Humana Press. https://doi.org/10.1007/978-1-61779-501-5_16
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DOI: https://doi.org/10.1007/978-1-61779-501-5_16
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