Agrobacterium-Mediated Insertional Mutagenesis in Histoplasma capsulatum
Genome-wide mutagenesis is a powerful method for identifying new genes that contribute to a phenotype of interest. For many fungal pathogens of plants and animals, Agrobacterium tumefaciens-mediated transformation (ATMT) serves as an efficient insertional
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1. Introduction Disruption of gene function is essential in defining the factors that contribute to the phenotype of an organism. For both forward and reverse genetics, success relies heavily on an efficient mutagen and, in a system where insertion occurs at random loci, the ability to identify the mutated locus. In the system described, the insertional mutagen imparts the advantage of tagging the mutation such that the nucleotide sequence flanking a random insertion can be rapidly determined and the disrupted gene identified. The plant pathogen Agrobacterium tumefaciens is capable of transforming many non-plant species through the transfer of a segment of DNA (transfer-DNA; T-DNA) from the bacterium into the host chromosome (1). Transfer of the T-DNA requires type IV
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_4, © Springer Science+Business Media, LLC 2012
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O. Zemska and C.A. Rappleye
pili contact between the bacterium and the target cell (2). A series of Vir proteins expressed by Agrobacterium initiate T-DNA transfer and production of these Vir proteins (and thus T-DNA transfer) is induced by phenolic compounds produced by wounded plant cells (3). The subsequent integration of transferred sequences into the target genome has led to the development of this system as an insertional mutagen for many fungi including Histoplasma capsulatum (4, 5). Agrobacterium bacteria harboring a T-DNA element engineered to carry an appropriate selection marker (e.g., hygromycin resistance) are cocultured with Histoplasma yeast and supplementation of the media with the vir gene stimulant, acetosyringone, induces the transfer functions (6, 7). After allowing sufficient time for transfer to occur, insertional mutants are obtained by moving the Agrobacterium-Histoplasma coculture to mediacontaining antibiotics to select for T-DNA-bearing cells and counterselect further growth of Agrobacterium. We describe here procedures for generating and identifying insertional mutants in Histoplasma yeast using Agrobacterium tumefaciens-mediated transformation (ATMT). The majority of T-DNA integrations into the Histoplasma genome are single copy (5) and the location of the chromosomal lesion can be determined by comparison of sequences flanking the integrated T-DNA element with the sequence of the Histoplasma genome. Inverse PCR, plasmid rescue, and thermal asymmetric interlaced PCR (TAIL-PCR) have all been successfully used to identify the genes disrupted by T-DNA insertion in Histoplasma (8–11). For TAIL-PCR, amplicons are anchored by a T-DNAspecific forward primer and a degenerate reverse primer that can bind in the region flanking the insertion site (12). Specific PCR products are obtained by alternating high-stringency and lowstringency cycles that allow the T-DNA-specific primer and the degenerate primer to bind, respectively (13). A secondary TAILPCR reaction is performed using the primary reaction as templat
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