Marschall Robert, Schumacher Julia, Siegmund Ulrike, Tudzynski Paul
Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität, Schlossplatz 8, D-48143 Münster, Germany.
Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität, Schlossplatz 8, D-48143 Münster, Germany.
Fungal Genet Biol. 2016 May;90:12-22. doi: 10.1016/j.fgb.2016.03.002. Epub 2016 Mar 14.
Reactive oxygen species (ROS) are important molecules influencing intracellular developmental processes as well as plant pathogen interactions. They are produced at the infection site and affect the intracellular redox homeostasis. However, knowledge of ROS signaling pathways, their connection to other signaling cascades, and tools for the visualization of intra- and extracellular ROS levels and their impact on the redox state are scarce. By using the genetically encoded biosensor roGFP2 we studied for the first time the differences between the redox states of the cytosol, the intermembrane space of mitochondria and the ER in the filamentous fungus Botrytis cinerea. We showed that the ratio of oxidized to reduced glutathione inside of the cellular compartments differ and that the addition of hydrogen peroxide (H2O2), calcium chloride (CaCl2) and the fluorescent dye calcofluor white (CFW) have a direct impact on the cellular redox states. Dependent on the type of stress agents applied, the redox states were affected in the different cellular compartments in a temporally shifted manner. By integrating the biosensor in deletion mutants of bcnoxA, bcnoxB, bctrx1 and bcltf1 we further elucidated the putative roles of the different proteins in distinct stress-response pathways. We showed that the redox states of ΔbcnoxA and ΔbcnoxB display a wild-type pattern upon exposure to H2O2, but appear to be strongly affected by CaCl2 and CFW. Moreover, we demonstrated the involvement of the light-responsive transcription factor BcLtf1 in the maintenance of the redox state in the intermembrane space of the mitochondria. Finally, we report that CaCl2 as well as cell wall stress-inducing agents stimulate ROS production and that ΔbcnoxB produces significantly less ROS than the wild type and ΔbcnoxA.
活性氧（ROS）是影响细胞内发育过程以及植物与病原体相互作用的重要分子。它们在感染部位产生，并影响细胞内的氧化还原稳态。然而，关于ROS信号通路、它们与其他信号级联的联系以及用于可视化细胞内和细胞外ROS水平及其对氧化还原状态影响的工具却很匮乏。通过使用基因编码的生物传感器roGFP2，我们首次研究了丝状真菌灰葡萄孢中细胞质、线粒体外膜间隙和内质网氧化还原状态之间的差异。我们发现细胞区室内部氧化型谷胱甘肽与还原型谷胱甘肽的比例不同，并且添加过氧化氢（H2O2）、氯化钙（CaCl2）和荧光染料荧光增白剂（CFW）会直接影响细胞的氧化还原状态。根据所施加应激剂的类型，不同细胞区室中的氧化还原状态会以时间上错开的方式受到影响。通过将生物传感器整合到bcnoxA、bcnoxB、bctrx1和bcltf1的缺失突变体中，我们进一步阐明了不同蛋白质在不同应激反应途径中的假定作用。我们发现，ΔbcnoxA和ΔbcnoxB在暴露于H2O2时呈现野生型模式，但似乎受到CaCl2和CFW的强烈影响。此外，我们证明了光响应转录因子BcLtf1参与维持线粒体外膜间隙的氧化还原状态。最后，我们报告CaCl2以及细胞壁应激诱导剂会刺激ROS的产生，并且ΔbcnoxB产生的ROS明显少于野生型和ΔbcnoxA。
