title: Construction and high-throughput phenotypic screening ofZymoseptoria tritici over-expression strains.
abstract: 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.
authors.affiliation: Biosciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK.
authors.forename: T C
authors.initials: TC
authors.lastname: Cairns
authors.affiliation: Biosciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK.
authors.forename: Y S
authors.initials: YS
authors.lastname: Sidhu
authors.affiliation: Biosciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK.
authors.forename: Y K
authors.initials: YK
authors.lastname: Chaudhari
authors.affiliation: Biosciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK.
authors.forename: N J
authors.initials: NJ
authors.lastname: Talbot
authors.affiliation: Biosciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK.
authors.forename: D J
authors.initials: DJ
authors.lastname: Studholme
authors.affiliation: Biosciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK. Electronic address: k.haynes@exeter.ac.uk.
authors.forename: K
authors.initials: K
authors.lastname: Haynes
mesh_terms: D001203:Ascomycota / Q000235:genetics*; D015870:Gene Expression; D018390:Gene Targeting / Q000379:methods*; D005820:Genetic Testing / Q000379:methods*; D057166:High-Throughput Screening Assays; D060847:Metabolic Engineering / Q000379:methods*
keywords: Functional genomics; High-throughput screen; Over-expression; Zymoseptoria tritici