Ghareeb Hassan, El-Sayed Mohamed, Pound Michael, Tetyuk Olena, Hanika Katharina, Herrfurth Cornelia, Feussner Ivo, Lipka Volker
Department of Plant Cell Biology, Albrecht-von-Haller Institute of Plant Sciences, University of Göttingen, Göttingen, Germany.
Department of Plant Biotechnology, National Research Centre, Cairo, Egypt.
Front Plant Sci. 2020 Nov 6;11:603693. doi: 10.3389/fpls.2020.603693. eCollection 2020.
The phytohormones salicylic acid (SA), jasmonic acid (JA), and ethylene (ET) are central regulators of biotic and abiotic stress responses in . Here, we generated modular fluorescent protein-based reporter lines termed COLORFUL-PR1pro, -VSP2pro, and -PDF1.2apro. These feature hormone-controlled nucleus-targeted transcriptional output sensors and the simultaneous constitutive expression of spectrally separated nuclear reference and plasma membrane-localized reporters. This set-up allowed the study of cell-type specific hormone activities, cellular viability and microbial invasion. Moreover, we developed a software-supported high-throughput confocal microscopy imaging protocol for output quantification to resolve the spatio-temporal dynamics of respective hormonal signaling activities at single-cell resolution. Proof-of-principle analyses in leaves revealed distinguished hormone sensitivities in mesophyll, epidermal pavement and stomatal guard cells, suggesting cell type-specific regulatory protein activities. In plant-microbe interaction studies, we found that virulent and avirulent () isolates exhibit different invasion dynamics and induce spatio-temporally distinct hormonal activity signatures. On the cellular level, these hormone-controlled reporter signatures demarcate the nascent sites of entry and progression, and highlight initiation, transduction and local containment of immune signals.
植物激素水杨酸（SA）、茉莉酸（JA）和乙烯（ET）是植物生物和非生物胁迫反应的核心调节因子。在此，我们构建了基于模块化荧光蛋白的报告株系，分别命名为COLORFUL-PR1pro、-VSP2pro和-PDF1.2apro。这些株系具有激素控制的细胞核靶向转录输出传感器，以及光谱分离的细胞核参考报告基因和质膜定位报告基因的同时组成型表达。这种设置使得对细胞类型特异性激素活性、细胞活力和微生物入侵的研究成为可能。此外，我们开发了一种软件支持的高通量共聚焦显微镜成像方案用于输出定量，以单细胞分辨率解析各自激素信号活性的时空动态。在拟南芥叶片中的原理验证分析揭示了叶肉细胞、表皮铺板细胞和气孔保卫细胞中不同的激素敏感性，表明存在细胞类型特异性的调节蛋白活性。在植物-微生物相互作用研究中，我们发现致病和无毒的丁香假单胞菌（Pseudomonas syringae）分离株表现出不同的入侵动态，并诱导时空上不同的激素活性特征。在细胞水平上，这些激素控制的报告特征划定了丁香假单胞菌进入和进展的新生部位，并突出了免疫信号的启动、转导和局部抑制。
