A codon-optimized green fluorescent protein for live cell imaging in Zymoseptoria tritici.
A codon-optimized green fluorescent protein for live cell imaging in Zymoseptoria tritici.
复制标题
DOI：
10.1016/j.fgb.2015.03.022
复制
发表时间：
2015-06
期刊：
影响因子：
--
通讯作者：
Steinberg G
中科院分区：
文献类型：
--
作者：
Kilaru S;Schuster M;Studholme D;Soanes D;Lin C;Talbot NJ;Steinberg G
我们生成了一个Z小麦密码子优化的绿色荧光蛋白（ZtGFP）基因。在落射荧光和共聚焦显微镜中，ZtGFP比eGFP更亮，更稳定。我们提供了3个载体，携带AcGFP，eGFP和ZtGFP的酵母重组为基础的克隆。所述载体携带针对靶向整合的羧菌素抗性。羧菌素抗性传递载体作为单拷贝整合到sdi 1基因座中。荧光蛋白是研究细胞内动力学和蛋白质定位的有力工具。FP在真菌病原体中的胞质表达允许更深入地了解入侵策略和宿主-病原体相互作用。其荧光信号的检测取决于显微镜设置和信号亮度的正确组合。缓慢的光漂白速率对于在较长时间段内体内观察FP是关键的。在这里，我们测试绿色荧光蛋白，包括蓝水母绿色荧光蛋白（AcGFP），增强型绿色荧光蛋白（eGFP）从维多利亚水母和一种新的Zymoseptoria三裂孔菌密码子优化的eGFP（ZtGFP），为他们的使用在传统的和激光增强落射荧光，共聚焦激光扫描显微镜。我们发现eGFP在Z. Trifecta，比AcGFP显著更亮且更光稳定。密码子优化的ZtGFP甚至比eGFP表现得更好，显示出明显更慢的漂白和20 - 30%的信号强度的进一步增加。所有GFP变异体的异源表达均不影响Z.三个月。我们的数据确立了ZtGFP作为研究Z细胞内蛋白质动力学的GFP选择。小麦病原菌在寄主组织内的侵染阶段。
We generated a Z. tritici codon-optimized gene for green fluorescent protein (ZtGFP). In epi-fluorescence and confocal microscopy, ZtGFP is brighter and more stable than eGFP. We provide 3 vectors that carry AcGFP, eGFP and ZtGFP for yeast recombination-based cloning. The vectors carry carboxin resistance for targeted integration. The carboxin resistance conveying vectors integrate as single copies into the sdi1 locus. Fluorescent proteins (FPs) are powerful tools to investigate intracellular dynamics and protein localization. Cytoplasmic expression of FPs in fungal pathogens allows greater insight into invasion strategies and the host-pathogen interaction. Detection of their fluorescent signal depends on the right combination of microscopic setup and signal brightness. Slow rates of photo-bleaching are pivotal for in vivo observation of FPs over longer periods of time. Here, we test green-fluorescent proteins, including Aequorea coerulescens GFP (AcGFP), enhanced GFP (eGFP) from Aequorea victoria and a novel Zymoseptoria tritici codon-optimized eGFP (ZtGFP), for their usage in conventional and laser-enhanced epi-fluorescence, and confocal laser-scanning microscopy. We show that eGFP, expressed cytoplasmically in Z. tritici, is significantly brighter and more photo-stable than AcGFP. The codon-optimized ZtGFP performed even better than eGFP, showing significantly slower bleaching and a 20–30% further increase in signal intensity. Heterologous expression of all GFP variants did not affect pathogenicity of Z. tritici. Our data establish ZtGFP as the GFP of choice to investigate intracellular protein dynamics in Z. tritici, but also infection stages of this wheat pathogen inside host tissue.