Department of Plant and Environmental Sciences, University of Copenhagen, Frederiksberg C, 1871, Denmark.
State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, Fujian Agriculture and Forestry University, Fuzhou, 35002, China.
Sci Rep. 2017 Mar 21;7(1):281. doi: 10.1038/s41598-017-00296-4.
Here, we report the development of a microplate reader-based system for visualizing gene expression dynamics in living bacterial cells in response to a fungus in space and real-time. A bacterium expressing the red fluorescent protein mCherry fused to the promoter region of a regulator gene nunF indicating activation of an antifungal secondary metabolite gene cluster was used as a reporter system. Time-lapse image recordings of the reporter red signal and a green signal from fluorescent metabolites combined with microbial growth measurements showed that nunF-regulated gene transcription is switched on when the bacterium enters the deceleration growth phase and upon physical encounter with fungal hyphae. This novel technique enables real-time live imaging of samples by time-series multi-channel automatic recordings using a microplate reader as both an incubator and image recorder of general use to researchers. The technique can aid in deciding when to destructively sample for other methods e.g. transcriptomics and mass spectrometry imaging to study gene expression and metabolites exchanged during the interaction.
在这里，我们报告了一种基于微孔板读数器的系统的开发，该系统可用于可视化活体细菌细胞对太空和实时真菌的基因表达动态。我们使用表达红色荧光蛋白 mCherry 融合到调控基因 nunF 启动子区域的细菌作为报告系统，该基因指示抗真菌次生代谢物基因簇的激活。报告红色信号和荧光代谢物的绿色信号的延时图像记录与微生物生长测量相结合，表明当细菌进入减速生长阶段并与真菌菌丝体物理接触时，nunF 调节的基因转录被打开。这项新技术通过使用微孔板读数器作为孵育器和图像记录器的通用多通道自动记录，实现了实时活体成像，为研究人员提供了便利。该技术可以帮助确定何时进行破坏性采样，以便使用其他方法（例如转录组学和质谱成像）来研究相互作用过程中基因表达和代谢物的交换。
