Targeted Gene Deletion in Aspergillus fumigatus Using the Hygromycin-Resistance Split-Marker Approach
The construction of a fungal strain that lacks a specific gene product is often accomplished by replacing the gene of interest with a selection marker using site-specific recombination. Transformation of Aspergillus fumigatus, like many related fungal spe
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1. Introduction Gene deletion is a powerful reverse-genetic approach for the study of gene function and virulence in filamentous fungi such as Aspergillus fumigatus. The most commonly used method for gene deletion is the replacement of a portion, or all, of the open reading frame (ORF) of interest with a selection marker. Although A. fumigatus auxotrophic markers such as pyrG have been used for transformation, dominant selection through the use of drug resistance markers is more common, and allows for the transformation of prototrophic strains. Two drug resistance markers are commonly used for selection of transformants: hph and ble. The hph cassette encodes hygromycin B phosphotransferase which confers resistance to hygromycin, while
Alexandra C. Brand and Donna M. MacCallum (eds.), Host-Fungus Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 845, DOI 10.1007/978-1-61779-539-8_8, © Springer Science+Business Media, LLC 2012
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ble encodes the Sh-ble protein that confers resistance to phleomycin. In this protocol, hph is used for the selection of gene disruption mutants, whereas ble is used for the subsequent selection of mutant strains that have been complemented by the reintegration of an intact copy of the wild-type allele of interest. Successful gene targeting in A. fumigatus requires overcoming two main difficulties: breaching the cell wall to allow entry of exogenous DNA and reducing ectopic nonhomologous recombination events (1). Here we present a detailed protocol for the use of protoplasting (2), combined with a hygromycin split-marker selection (3) for the deletion of A. fumigatus genes that has been successful in overcoming these challenges. Protoplasting utilizes a cocktail of enzymes to digest the carbohydrate-rich cell wall of A. fumigatus, thereby releasing membrane bound protoplasts, some of which contain intact nuclei. These protoplasts are then mixed with the transforming DNA, which can bind to the protoplast plasma membrane. Heat shock is thought to induce a localized inversion of the DNA–membrane complex, resulting in internalization of the extracellular DNA and subsequent recombination (2). The use of a split-marker approach greatly improves the recovery of homologous integrants. Briefly, two hybrid DNA constructs are used for cotransformation, each comprising a fusion product of one of the flanking sequences of the targeted gene with an incomplete portion of the drug resistance cassette (Fig. 1). These drug resistance sequences overlap for about 600 bp. Neither marker fragment encodes sufficient sequences to reconstitute the functional resistance protein. Therefore, acquisition of drug resistance requires homologous recombination of the marker halves within their overlapping sequences. The highest probability for this event occurs when the fragment DNAs are in proximity to one another, such as during integration at the target locus. This promotes a triple recombination event which replaces the target gene and reconstitutes the functional dr
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