Department of Plant Sciences, The University of Tennessee, Knoxville, 37996, USA.
BMC Biotechnol. 2011 Nov 17;11:108. doi: 10.1186/1472-6750-11-108.
We aimed to engineer transgenic plants for the purpose of early detection of plant pathogen infection, which was accomplished by employing synthetic pathogen inducible promoters fused to reporter genes for altered phenotypes in response to the pathogen infection. Toward this end, a number of synthetic promoters consisting of inducible regulatory elements fused to a red fluorescent protein (RFP) reporter were constructed for use in phytosensing.
For rapid analysis, an Agrobacterium-mediated transient expression assay was evaluated, then utilized to assess the inducibility of each synthetic promoter construct in vivo. Tobacco (Nicotiana tabacum cv. Xanthi) leaves were infiltrated with Agrobacterium harboring the individual synthetic promoter-reporter constructs. The infiltrated tobacco leaves were re-infiltrated with biotic (bacterial pathogens) or abiotic (plant defense signal molecules salicylic acid, ethylene and methyl jasmonate) agents 24 and 48 hours after initial agroinfiltration, followed by RFP measurements at relevant time points after treatment. These analyses indicated that the synthetic promoter constructs were capable of conferring the inducibility of the RFP reporter in response to appropriate phytohormones and bacterial pathogens, accordingly.
These observations demonstrate that the Agrobacterium-mediated transient expression is an efficient method for in vivo assays of promoter constructs in less than one week. Our results provide the opportunity to gain further insights into the versatility of the expression system as a potential tool for high-throughput in planta expression screening prior to generating stably transgenic plants for pathogen phytosensing. This system could also be utilized for temporary phytosensing; e.g., not requiring stably transgenic plants.
我们旨在通过将合成病原体诱导启动子与报告基因融合，以改变对病原体感染的表型，从而为植物病原体感染的早期检测工程转基因植物。为此，构建了许多由诱导性调控元件与红色荧光蛋白（RFP）报告基因融合的合成启动子，用于植物感应。
为了快速分析，评估了农杆菌介导的瞬时表达测定法，然后用于体内评估每个合成启动子构建体的诱导性。用携带单个合成启动子-报告基因构建体的农杆菌浸润烟草（Nicotiana tabacum cv. Xanthi）叶片。用生物（细菌病原体）或非生物（植物防御信号分子水杨酸、乙烯和茉莉酸甲酯）试剂在初始农杆菌浸润后 24 和 48 小时再次浸润浸润的烟草叶片，然后在处理后相关时间点测量 RFP。这些分析表明，合成启动子构建体能够赋予 RFP 报告基因对适当植物激素和细菌病原体的诱导性。
这些观察结果表明，农杆菌介导的瞬时表达是在不到一周的时间内对启动子构建体进行体内分析的有效方法。我们的结果为进一步了解表达系统的多功能性提供了机会，作为在生成用于病原体植物感应的稳定转基因植物之前进行高通量植物体内表达筛选的潜在工具。该系统还可用于临时植物感应；例如，不需要稳定的转基因植物。
