Construction and high-throughput phenotypic screening ofZymoseptoria tritici over-expression strains.
Construction and high-throughput phenotypic screening ofZymoseptoria tritici over-expression strains.
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DOI：
10.1016/j.fgb.2015.04.013
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发表时间：
2015-06
期刊：
影响因子：
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通讯作者：
Haynes K
中科院分区：
文献类型：
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作者：
Cairns TC;Sidhu YS;Chaudhari YK;Talbot NJ;Studholme DJ;Haynes K
我们建立了32株小麦偃麦草过表达菌株的先导文库。我们开发了一种高通量的体外表型筛选技术。该协议可以探测小麦小麦菌丝和出芽的形态。一个假定的转录因子在过度表达时阻碍菌丝的生物合成。全基因组功能分析将有助于发现新的毒力属性。靶向基因缺失有助于阐明小麦酵母致病性的许多方面。基因过表达是一种互补的方法，适用于快速菌株构建和高通量筛选，但由于缺乏可用的技术，尚未被用于分析小麦弧菌。本研究利用Gateway®克隆技术快速构建小麦偃麦草Δku70过表达载体并提高同源整合效率，构建了包含32个编码DNA结合蛋白、gtpase或激酶的基因的中试过表达文库。我们开发了一种使用Rotor-HDA机器人的方案，用于快速和可重复的细胞固定，用于高通量体外筛选。该筛选鉴定出一种过表达菌株，与等基因祖细胞相比，其菌丝产量明显减少。本研究为快速生成小麦偃麦草过表达文库提供了一种方案，并为这一重要病原菌的功能基因组筛选提供了技术手段。
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.