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

Simon Uhse,Arndt Von Haeseler,Klaus Ehrlinger,Alexandra Stirnberg,Florian G Pflug,Armin Djamei,Joseph Heitman

doi:10.1371/journal.pbio.2005129

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.

Highlights

Virulence factors are key for successful infections by pathogens
A major limitation is the efficient identification of mutants that are lost or retained after selection
The two genomic sequences flanking the insertion cassette must be found. Pinpointing these insertion flanks within a genome is like looking for a needle in a haystack; a problem that becomes even worse when several organisms form a biotrophic interaction

Summary

Introduction

Virulence factors are key for successful infections by pathogens. Their identification is of major interest because of the necessity to develop effective counter strategies. Fungal virulence factors are typically identified by mutating single loci in fungi, followed by individual fungal mutant infections of host tissue and subsequent assessment of pathogen fitness [1,2,3,4]. An attractive alternative is infection with a pool of pathogen mutants allowing direct assessment of individual pathogen fitness in the same host tissue. Using a pooled pathogen infection creates the challenge of identifying pathogens with reduced virulence within a complex mixture of genetic material extracted from infected host tissue

Methods

Results

Discussion

Conclusion