In vivo insertion pool sequencing identifies virulence factors in a complex fungal–host interaction

<div><p>Large-scale insertional mutagenesis screens can be powerful genome-wide tools if they are streamlined with efficient downstream analysis, which is a serious bottleneck in complex biological systems. A major impediment to the success of next-generation sequencing (NGS)-based screens for virulence factors is that the genetic material of pathogens is often underrepresented within the eukaryotic host, making detection extremely challenging. We therefore established insertion Pool-Sequencing (iPool-Seq) on maize infected with the biotrophic fungus <i>U</i>. <i>maydis</i>. iPool-Seq features tagmentation, unique molecular barcodes, and affinity purification of pathogen insertion mutant DNA from in vivo-infected tissues. In a proof of concept using iPool-Seq, we identified 28 virulence factors, including 23 that were previously uncharacterized, from an initial pool of 195 candidate effector mutants. Because of its sensitivity and quantitative nature, iPool-Seq can be applied to any insertional mutagenesis library and is especially suitable for genetically complex setups like pooled infections of eukaryotic hosts.</p></div

iPool-Seq library preparation workflow features tagmentation and UMIs.

<p>(<b>a</b>) Library preparation was carried out for the input mutant collection and for the output after infection. For the output, we harvested infected areas of the second and third maize leaves and isolated gDNA. (<b>b</b>) Extracted gDNA was fragmented with Tn5 Transposase loaded with custom adapters containing an SBS (green), 12-bp UMI, and Tn5 hyperactive MEs (blue). Genome–hpt resistance cassette junctions were PCR-amplified with biotinylated primers directed against UPSs (magenta) and adapter-specific primers directed at the SBS. (<b>c</b>) Biotinylated PCR products were streptavidin-affinity–purified and Illumina-compatible P5 (purple; NGS1) and P7 (purple; NGS2) ends were introduced by nested PCR. Final products were subjected to Illumina PE sequencing on a MiSeq platform. gDNA, genomic DNA; hpt, hygromycin phosphotransferase; iPool-Seq, insertion Pool-Sequencing ME, mosaic end; PE, paired-end; ROI, region of interest; SBS, sequencing primer binding site; UMI, unique molecular identifier; UPS, unique primer binding site.</p