Plant Fungal Pathogens Methods and Protocols
Over the course of evolution, fungi have adapted to occupy specific niches, from symbiotically inhabiting the flora of the intestinal tract of mammals to saprophytic growth on leaf litter resting on the forest floor. In Plant Fungal Pathogens: Metho
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1. Introduction Gene replacement has been one of the fundamental approaches used for functional characterization of fungal genes or open reading frames. Over the years, several techniques have been developed and adopted for this purpose. With the increased availability of whole genome sequences, the split-marker deletion approach is becoming more widely adopted for functional genomic studies.
Melvin D. Bolton and Bart P.H.J. Thomma (eds.), Plant Fungal Pathogens: Methods and Protocols, Methods in Molecular Biology, vol. 835, DOI 10.1007/978-1-61779-501-5_16, © Springer Science+Business Media, LLC 2012
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This approach relies on the availability of the sequence of the gene of interest and portions flanking it on both sides prior to replacement. This strategy was initially developed for Saccharomyces cerevisiae by Fairhead et al. (1, 2) and is reported to have been used for Cochliobolus heterostrophus and Gibberella zeae (3, 4). Split-marker is a PCR-based method that does not require subcloning and only involves two rounds of PCR. The hygromycin B phosphotransferase (hph) gene conferring resistance to the antibiotic hygromycin or any other marker is used, and it depends upon homologous recombination for gene replacement in the fungus. The technique involves PCR amplification of regions flanking the gene of interest on either side 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 (hph) gene fused to one flanking region. These two cassettes are simultaneously used for transformation. Three homologous recombination events occur, one within each flanking region and one in the marker (hph) gene, to successfully replace the gene of interest with a functional marker (hph) gene. The methods described here were developed for Fusarium graminearum and the hph gene as a selectable marker; however, the marker can be easily changed. Modifications that may be required based on the fungal species being transformed, source construct for the marker gene, and other factors are mentioned in the text.
2. Materials Solutions, specific media, supplies, and specific equipment used at various stages of the protocol: 2.1. DNA Extraction
1. Nuclei Lysis Buffer: 100 mL 1 M Tris pH 7.5, 50 mL of 0.5 M EDTA pH 8, 10 g of Cetyl Trimethyl Ammonium Bromide (CTAB) are added to 500 mL with distilled water and autoclaved. 2. DNA Isolation Buffer: 31.89 g Sorbitol, 6.05 g Tris Base/ THAM, and 0.84 g EDTA are added to 500 mL distilled water, the pH is adjusted to 7.5 and autoclaved. 3. 5% Sarkosyl: 12.5 g N-laurylsarkosine is added to 250 mL distilled water and autoclaved. 4. DNA extraction buffer: Mix 1:1:0.4 volume of the Nuclei lysis buffer, DNA isolation buffer, and 5% Sarkosyl to prepare this buffer. 5. TE (pH 8): 4.0 mL of 1 M Tris pH 8 and 0.8 mL of 0.5 M EDTA pH 8 are added to 400 mL distilled water and autoclaved. 6. Complete medium: Vitamin and trace element stock solutions need to be prepared first. The vitamin stock solution is co
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