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

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

Abstract

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.

Grafik Top
Authors
  • Uhse, Simon
  • Pflug, Florian G.
  • Stirnberg, Alexandra
  • Ehrlinger, Klaus
  • von Haeseler, Arndt
  • Djamei, Armin
Grafik Top
Shortfacts
Category
Journal Paper
Divisions
Bioinformatics and Computational Biology
Journal or Publication Title
PLoS Biology
ISSN
1544-9173
Publisher
PLoS
Place of Publication
San Francisco, California, US PLOS
Page Range
e2005129
Number
4
Volume
16
Date
23 April 2018
Official URL
http://dx.doi.org/10.1371/journal.pbio.2005129
Export
Grafik Top