Persad-Russell Ramona, Mazarei Mitra, Schimel Tayler Marie, Howe Lana, Schmid Manuel J, Kakeshpour Tayebeh, Barnes Caitlin N, Brabazon Holly, Seaberry Erin M, Reuter D Nikki, Lenaghan Scott C, Stewart C Neal
Department of Plant Sciences, The University of Tennessee, Knoxville, Knoxville, TN, United States.
Center for Agricultural Synthetic Biology, The University of Tennessee, Knoxville, Knoxville, TN, United States.
Front Plant Sci. 2022 Apr 25;13:873480. doi: 10.3389/fpls.2022.873480. eCollection 2022.
Phytosensors are genetically engineered plant-based sensors that feature synthetic promoters fused to reporter genes to sense and report the presence of specific biotic and abiotic stressors on plants. However, when induced reporter gene output is below detectable limits, owing to relatively weak promoters, the phytosensor may not function as intended. Here, we show modifications to the system to amplify reporter gene signal by using a synthetic transcription factor gene driven by a plant pathogen-inducible synthetic promoter. The output signal was unambiguous green fluorescence when plants were infected by pathogenic bacteria. We produced and characterized a phytosensor with improved sensing to specific bacterial pathogens with targeted detection using spectral wavelengths specific to a fluorescence reporter at 3 m standoff detection. Previous attempts to create phytosensors revealed limitations in using innate plant promoters with low-inducible activity since they are not sufficient to produce a strong detectable fluorescence signal for standoff detection. To address this, we designed a pathogen-specific phytosensor using a synthetic promoter-transcription factor system: the S-Box -regulatory element which has low-inducible activity as a synthetic 4xS-Box promoter, and the Q-system transcription factor as an amplifier of reporter gene expression. This promoter-transcription factor system resulted in 6-fold amplification of the fluorescence after infection with a potato pathogen, which was detectable as early as 24 h post-bacterial infection. This novel bacterial pathogen-specific phytosensor potato plant demonstrates that the Q-system may be leveraged as a powerful orthogonal tool to amplify a relatively weak synthetic inducible promoter, enabling standoff detection of a previously undetectable fluorescence signal. Pathogen-specific phytosensors would be an important asset for real-time early detection of plant pathogens prior to the display of disease symptoms on crop plants.
植物传感器是基于基因工程的植物型传感器，其特点是将合成启动子与报告基因融合，以感知并报告植物上特定生物和非生物胁迫因子的存在。然而，由于启动子相对较弱，当诱导的报告基因输出低于可检测限时，植物传感器可能无法按预期发挥作用。在此，我们展示了对该系统的改进，即通过使用由植物病原体诱导型合成启动子驱动的合成转录因子基因来放大报告基因信号。当植物受到病原菌感染时，输出信号为明确的绿色荧光。我们制备并表征了一种植物传感器，该传感器通过在3米远距离检测时使用荧光报告基因特有的光谱波长进行靶向检测，对特定细菌病原体具有更好的传感能力。以往创建植物传感器的尝试揭示了使用具有低诱导活性的天然植物启动子存在局限性，因为它们不足以产生用于远距离检测的强可检测荧光信号。为了解决这个问题，我们使用合成启动子 - 转录因子系统设计了一种病原体特异性植物传感器：作为合成4xS - Box启动子具有低诱导活性的S - Box调控元件，以及作为报告基因表达放大器的Q - 系统转录因子。这种启动子 - 转录因子系统在感染马铃薯病原体后使荧光放大了6倍，早在细菌感染后24小时就可检测到。这种新型的细菌病原体特异性植物传感器马铃薯植株表明，Q - 系统可作为一种强大的正交工具来放大相对较弱的合成诱导型启动子，从而实现对先前无法检测到的荧光信号的远距离检测。病原体特异性植物传感器将是在作物植株出现疾病症状之前实时早期检测植物病原体的一项重要资产。
