Construction and high-throughput phenotypic screening ofZymoseptoria tritici over-expression strains

•We generate a pilot library of 32 Z. tritici over-expression strains.•We develop a high throughput technique for in vitro phenotypic screening.•This protocol can probe both hyphal and budding Z. tritici morphologies.•A putative transcription factor impedes hyphal biosynthesis when over-expressed.•Genome wide functional analysis will enable discovery of novel virulence attributes. Targeted gene deletion has been instrumental in elucidating many aspects of Zymoseptoria tritici pathogenicity. Gene over-expression is a complementary approach that is amenable to rapid strain construction and high-throughput screening, which has not been exploited to analyze Z. tritici, largely due to a lack of available techniques. Here we exploit the Gateway® cloning technology for rapid construction of over-expression vectors and improved homologous integration efficiency of a Z. tritici Δku70 strain to build a pilot over-expression library encompassing 32 genes encoding putative DNA binding proteins, GTPases or kinases. We developed a protocol using a Rotor-HDA robot for rapid and reproducible cell pinning for high-throughput in vitro screening. This screen identified an over-expression strain that demonstrated a marked reduction in hyphal production relative to the isogenic progenitor. This study provides a protocol for rapid generation of Z. tritici over-expression libraries and a technique for functional genomic screening in this important pathogen.