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.

Insertion Pool Sequencing for Insertional Mutant Analysis in Complex Host‐Microbe Interactions

“…However, tools that allow for efficient high‐throughput analysis of a negative depletion screen in the context of a host, for instance in the case of M. oryzae and rice (Jeon et al., ), were not available until recently. Therefore, we developed iPool‐Seq, which, due to its high selectivity and sensitivity, allows for analysis of pooled infections of insertional mutants directly from the infected host tissue (Uhse et al., ). iPool‐Seq provides a powerful tool for scientists who want to analyze mutant pool composition after colonization of the host, without any biases that could arise due to separation of the output fraction.…”

“…We previously demonstrated (Uhse et al., ) that iPool‐Seq offers an elegant possibility to analyze a U. maydis insertional mutant pool after colonization of its host maize. We further suggest that iPool‐Seq could be applied to genome‐wide insertional mutant pools generated by high‐throughput techniques, e.g., transposon‐mediated mutagenesis.…”

“…The confirmation of these known virulence factors by iPool‐Seq indicates that iPool‐Seq yields reliable results for depleted mutants. To further strengthen this finding, we tested three potential virulence factors identified by iPool‐Seq and were able to show a virulence phenotype for these mutants based on classical disease ratings in comparison to the wildtype solopathogenic strain SG200 (Uhse et al., ).…”

“…Recent advances in massive parallel sequencing allow for large‐scale approaches in bacteria that permit analysis of larger pools of insertional mutants (Gawronski, Wong, Giannoukos, Ward, & Akerley, ; van Opijnen, Bodi, & Camilli, ). Here, we describe the insertion pool sequencing (iPool‐Seq) pipeline that we recently established with the Ustilago maydis – Zea mays interaction (Uhse et al., ).…”

“…We created an insertional mutant library for U. maydis via homologous recombination of a selectable marker conferring resistance to hygromycin. Next, we established the iPool‐Seq workflow based on this library, allowing for controlled insertional mutagenesis at loci of predicted effectors that are likely to contribute to the virulence of U. maydis (Uhse et al., ). All newly generated U. maydis mutants were verified for deletion of the targeted effector genes via PCR on cultures and on extracted genomic DNA (gDNA).…”