Spectrum of T-DNA integrations for insertional mutagenesis of Histoplasma capsulatum

Abstract

Agrobacterium-mediated transformation is being increasingly used for insertional mutagenesis of fungi. To better evaluate its effectiveness as a mutagen for the fungal pathogen Histoplasma capsulatum, we analyzed a collection of randomly selected T-DNA insertion mutants. Testing of different T-DNA element vectors engineered for transformation of fungi showed that pBHt2 provides the highest transformation efficiency and the lowest rate of vector backbone carryover. Sixty-eight individual T-DNA integrations were characterized by recovery of T-DNA ends and flanking genomic sequences. The right border end of the T-DNA is largely preserved whereas the left border end is frequently truncated. Analysis of T-DNA insertion sites confirms the lack of any integration hotspots in the Histoplasma genome. Relative to genes, T-DNA integrations show significant bias towards promoter regions at the expense of coding sequences. With consideration for potential promoter interruption and the demonstrated efficacy of intronic insertions, 61% of mapped T-DNA insertions should impair gene expression or function. Mapping of T-DNA flanking sequences demonstrates 67% of T-DNA integrations are integrations at a single chromosomal site and 31% of T-DNA integrations are associated with large-scale chromosomal rearrangements. This characterization of T-DNA insertions in mutants selected without regard to phenotype supports application of Agrobacterium-mediated transformation as an insertional mutagen for genome-based screens and functional discovery of genes in Histoplasma.

Rapid mapping of insertional mutations to probe cell wall regulation in Cryptococcus neoformans

“…The remaining two events involved larger insert-induced deletions, including one deletion of 1.2 kb (Figure 2C). Large deletions have been described in other fungi, however they are thought to occur uncommonly (Kemski et al, 2013; Li et al, 2007; Meng et al, 2007). …”

Histoplasma capsulatum Depends on De Novo Vitamin Biosynthesis for Intraphagosomal Proliferation

“…Histoplasma yeasts were mutagenized using Agrobacterium-mediated transformation (47)(48)(49). Agrobacterium tumefaciens strain LBA1100 harboring plasmid pCM41 was cocultured with WU15 Histoplasma yeasts for 48 h and transferred to HMM plus uracil medium containing 100 g/ml hygromycin to select for Histoplasma transformants.…”

“…The location of the T-DNA insertion was determined by thermal asymmetric interlaced PCR (TAIL-PCR) (47)(48)(49)(50). Two hundred nanograms of genomic DNA was used as the template for the primary PCR, with a T-DNA left border (LB)-or right border (RB)-specific primer (LB11 or RB9) and one of four semirandom primers (LAD1 to -4).…”

pFPL Vectors for High-Throughput Protein Localization in Fungi: Detecting Cytoplasmic Accumulation of Putative Effector Proteins

“…Recognizing the power of Gateway TM cloning for high-throughput functional studies in plants, Goodin and coworkers generated a comprehensive collection of Gateway TM cloning vectors (Chakrabarty et al, 2007) that are now in wide use among the plant research community (Jeong et al, 2010;Selote and Kachroo, 2010;Dietzgen et al, 2012;Levy et al, 2013). To facilitate our screening for effector candidates, we utilized the Gateway TM fluorescent protein (FP) cassettes from the pSITE-C vector series and incorporated them into the binary vector, pBHt2, which is commonly used for efficient Agrobacterium tumefaciens-mediated transformation (ATMT) of fungi (Rho et al, 2001;Nyilasi et al, 2005;Talhinhas et al, 2008;Jackson et al, 2009;Rolland et al, 2009;Kemski et al, 2013).…”