Fraiture Malou, Zheng Xiangzi, Brunner Frédéric
Department of Plant Biochemistry, Centre for Plant Molecular Biology, Eberhard Karls University, Auf der Morgenstelle 32, 72076, Tübingen, Germany.
Methods Mol Biol. 2014;1127:213-30. doi: 10.1007/978-1-62703-986-4_17.
Transient expression in plant mesophyll protoplasts allows rapid characterisation of gene functions in vivo in a simplified and synchronized manner without bias due to the use of bacteria-based gene or protein delivery systems. It offers the possibility to test whether microbial effectors can subvert early events of plant immune signaling that are activated upon recognition of Microbe-Associated Molecular Patterns (MAMPs), the so-called MAMP-triggered immunity (MTI). Here, we describe the isolation and transfection with effector genes of Arabidopsis thaliana and Solanum lycopersicum mesophyll protoplasts, the use of a non-invasive luciferase reporter assay and a simple method to detect activated Mitogen-Activated Protein Kinases (MAPKs) to identify and study, in a medium-throughput manner, new effectors suppressing early signal transduction events of MTI.
植物叶肉原生质体中的瞬时表达能够以简化且同步的方式在体内快速表征基因功能，且不会因使用基于细菌的基因或蛋白质递送系统而产生偏差。它提供了一种可能性，即测试微生物效应子是否能够颠覆植物免疫信号传导的早期事件，这些事件在识别微生物相关分子模式（MAMPs）时被激活，即所谓的MAMP触发免疫（MTI）。在此，我们描述了拟南芥和番茄叶肉原生质体效应子基因的分离与转染、非侵入性荧光素酶报告基因检测的使用以及一种检测活化丝裂原活化蛋白激酶（MAPKs）的简单方法，以便以中等通量的方式鉴定和研究抑制MTI早期信号转导事件的新效应子。
