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Fungal Genet Biol. 2015 Jun;79:110-7. doi: 10.1016/j.fgb.2015.04.013.

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

Author information

1
Biosciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK.
2
Biosciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK. Electronic address: k.haynes@exeter.ac.uk.

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.

KEYWORDS:

Functional genomics; High-throughput screen; Over-expression; Zymoseptoria tritici

PMID:
26092797
PMCID:
PMC4502453
DOI:
10.1016/j.fgb.2015.04.013
[Indexed for MEDLINE]
Free PMC Article

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