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Construction and high-throughput phenotypic screening of Zymoseptoria tritici over-expression strains

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 du...

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Detalles Bibliográficos
Autores principales: Cairns, T.C., Sidhu, Y.S., Chaudhari, Y.K., Talbot, N.J., Studholme, D.J., Haynes, K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Academic Press 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4502453/
https://www.ncbi.nlm.nih.gov/pubmed/26092797
http://dx.doi.org/10.1016/j.fgb.2015.04.013
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author Cairns, T.C.
Sidhu, Y.S.
Chaudhari, Y.K.
Talbot, N.J.
Studholme, D.J.
Haynes, K.
author_facet Cairns, T.C.
Sidhu, Y.S.
Chaudhari, Y.K.
Talbot, N.J.
Studholme, D.J.
Haynes, K.
author_sort Cairns, T.C.
collection PubMed
description 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.
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spelling pubmed-45024532015-07-21 Construction and high-throughput phenotypic screening of Zymoseptoria tritici over-expression strains Cairns, T.C. Sidhu, Y.S. Chaudhari, Y.K. Talbot, N.J. Studholme, D.J. Haynes, K. Fungal Genet Biol Article 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. Academic Press 2015-06 /pmc/articles/PMC4502453/ /pubmed/26092797 http://dx.doi.org/10.1016/j.fgb.2015.04.013 Text en © 2015 The Authors http://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Cairns, T.C.
Sidhu, Y.S.
Chaudhari, Y.K.
Talbot, N.J.
Studholme, D.J.
Haynes, K.
Construction and high-throughput phenotypic screening of Zymoseptoria tritici over-expression strains
title Construction and high-throughput phenotypic screening of Zymoseptoria tritici over-expression strains
title_full Construction and high-throughput phenotypic screening of Zymoseptoria tritici over-expression strains
title_fullStr Construction and high-throughput phenotypic screening of Zymoseptoria tritici over-expression strains
title_full_unstemmed Construction and high-throughput phenotypic screening of Zymoseptoria tritici over-expression strains
title_short Construction and high-throughput phenotypic screening of Zymoseptoria tritici over-expression strains
title_sort construction and high-throughput phenotypic screening of zymoseptoria tritici over-expression strains
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4502453/
https://www.ncbi.nlm.nih.gov/pubmed/26092797
http://dx.doi.org/10.1016/j.fgb.2015.04.013
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