In vivo insertion pool sequencing identifies virulence factors in a complex fungal-host interaction.
Simon UhseFlorian G PflugAlexandra StirnbergKlaus EhrlingerArndt von HaeselerArmin DjameiPublished in: PLoS biology (2018)
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 U. maydis. 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.
Keyphrases
- genome wide
- dna methylation
- copy number
- antimicrobial resistance
- pseudomonas aeruginosa
- escherichia coli
- staphylococcus aureus
- single cell
- biofilm formation
- crispr cas
- gene expression
- circulating tumor
- rna seq
- randomized controlled trial
- regulatory t cells
- candida albicans
- high throughput
- dendritic cells
- cystic fibrosis
- clinical trial
- gram negative
- multidrug resistant
- mass spectrometry
- phase iii