Insertion Pool Sequencing Identifies Essential Fungal Virulence Factors in vivo

Large scale insertion mutant screens are powerful tools in research, but have limitations regarding complex biological systems. In pathosystems, insertional mutants can help to identify virulence factors of the pathogen. However, pathogens are often highly underrepresented after infection of their multicellular eukaryotic host, which makes it difficult to extract sufficient amounts for downstream analysis. To overcome this bottleneck, we established insertion Pool-Sequencing (iPool-Seq) with a mutant collection of the biotrophic plant fungus Ustilago maydis. iPool-Seq library preparation features tagmentation and unique molecular barcodes to enrich efficiently for insertional mutant cassettes out of in vivo infected maize host tissue. We generated high quality NGS libraries and identified 5 known and 19 novel fungal virulence factors. Moreover, we confirmed the virulence defect of three novel candidates indicating that iPool-Seq works reliable. We suggest, that due to its sensitive and quantitative characteristics iPool-Seq can be applied in any insertional mutagenesis library and is especially suitable for complex setups like pooled infections on eukaryotic hosts.