| Highly Accurate Real-time Measurement of Rapid Hydrogen-peroxide Dynamics in Fungi
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Author:
Date:
2016-12-20
[Abstract] Reactive oxygen species (ROS) are unavoidable by-products of aerobic metabolism. Despite beneficial aspects as a signaling molecule, ROS are principally recognized as harmful agents that act on nucleic acids, proteins and lipids. Reactive oxygen species, and, in particular, hydrogen peroxide (H2O2), are deployed as defense molecules across kingdoms, e.g., by plants in order to defeat invading pathogens like fungi. Necrotrophic plant pathogenic fungi themselves secrete H2O2 to induce host cell death and facilitate infection. Hydrogen peroxide is, to a certain extent, freely diffusible through membranes. To be able to monitor intracellular hydrogen peroxide dynamics in fungi, we recently established the versatile HyPer-imaging technique ...
[摘要] 活性氧（ROS）是有氧代谢的副产物。尽管作为信号分子的有利方面，ROS主要被认为是作用于核酸，蛋白质和脂质的有害物质。反应性氧物质，特别是过氧化氢（H 2 O 2 O 2），作为防御分子跨越诸如，通过植物为了击败入侵病原体如真菌。营养不良的植物致病真菌本身分泌H 2 O 2 O 2以诱导宿主细胞死亡并促进感染。过氧化氢在一定程度上可以通过膜自由扩散。为了能够监测真菌中的细胞内过氧化氢动力学，我们最近在丝状植物病原体禾谷镰刀菌（Mentges and Bormann，2015）中建立了多功能的HyPer成像技术。 HyPer由插入到原核H 2 O 2 O 2 - 感觉蛋白OxyR的调节结构域（RD）中的循环置换的黄色荧光蛋白（cpYFP）组成。 OxyR域使传感器高度特异于H 2 O 2 O 2。 HyPer的氧化增加了在488nm激发的cpYFP的荧光，并降低了在405nm激发的荧光，从而促进了比例式读数（Belousov等人，2006）。 HyPer原来是对pH敏感。 HyPer的H 2 O 2 O 2 - 感官域中的单个氨基酸突变使传感器对H 2 O 2不敏感。该记者称为SypHer，作为pH变化的对照。
＆nbsp; ＆nbsp;通过使用HyPer成像技术，我们可以证明：i）HyPer成像能够特异和准确地检测细胞内H 2 O 2 O 2平衡的快速变化，ii）。 ...
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| Ustilago maydis Virulence Assays in Maize
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Author:
Date:
2016-03-20
[Abstract] The basidiomycetous smut fungus Ustilago maydis (U. maydis) infects all aerial parts of its host plant maize (Zea mays L.). Infection symptoms are seen in the form of prominent tumors on all aerial parts of maize, after the establishment of a biotrophic interaction with the host usually around 5-6 days post infection (dpi). The fungus colonizes the various developmentally distinct aerial organs at different stages of development to form these prominent symptoms. Although being a biotrophic plant pathogen, U. maydis can easily be cultivated under axenic conditions to produce a standardized inoculum. The infections can be carried out under laboratory conditions by syringe inoculation on all the aerial organs of maize. This protocol has been successfully ...
[摘要] 落叶青霉菌（Basidiomycetous smut fungus）Ustilago maydis（ U。maydis ）感染其宿主植物玉米的所有地上部分（ Zea mays 感染症状在与宿主生物营养相互作用通常在感染后约5-6天（dpi）建立后以玉米的所有地上部分的突出肿瘤的形式见到。真菌在不同发育阶段定居各种发育不同的气生器官以形成这些突出的症状。虽然是一种生物营养性植物病原体， maydis 可以容易地在无菌条件下培养以产生标准化接种物。感染可以在实验室条件下通过注射器接种在玉米的所有气生器官上进行。该方案已经成功地用于感染玉米的所有气生器官并且在U中制定毒力测定。 maydis 使其成为研究植物病理学研究的优良模型系统（Schilling等人，2014; Redkar等人，2015）。
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| Localization and Topology of Thylakoid Membrane Proteins in Land Plants
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Author:
Date:
2014-12-20
[Abstract] Thylakoids are a formation of flattened membrane vesicles and protein complexes found in cyanobacteria, algae and plants. In the chloroplasts of land plants the thylakoid membrane systems form a network of densely packed stacks called grana lamellae, which are connected by unstacked stroma lamellae. Photosystem II is mainly localized in the appressed grana region, while photosystem I and the ATP synthase complexes are enriched in the stroma lamellae. The cytochrome b6/f complex is distributed laterally throughout both stacked and unstacked membrane regions. The photosynthetic complexes consist of integral and peripheral proteins. The first part of this protocol (A) shows how to fractionate thylakoids into grana and stroma lamellae. The second part of this ...
[摘要] 类囊体是在蓝细菌，藻类和植物中发现的扁平膜囊泡和蛋白质复合物的形成。在陆地植物的叶绿体中，类囊体膜系统形成称为颗粒薄片的密集堆叠的网络，其通过未堆叠的基质薄片连接。光系统II主要定位在贴壁的颗粒区域，而光系统I和ATP合酶复合物富集基质层。细胞色素e / f 复合物横向分布在堆叠和未堆叠的膜区域。光合复合物由整合蛋白和外周蛋白组成。本协议（A）的第一部分显示如何分裂类囊体成grana和基质层。该方案的第二部分（B）显示了如何区分强疏水整合膜缔合和弱静电膜和/或膜复合物缔合。由于必须特异性检测级分中的目标蛋白，针对目的蛋白产生的特异性抗体或表达标记融合蛋白的结构组分的互补无效突变体将是非常有利的。本协议（C）的最后一部分显示，如何调查内在和外围蛋白的拓扑。该方法需要针对目标蛋白的特异性抗体。对于内在膜蛋白，需要肽特异性抗体或表位标记的形式。该方案适合于低于5kDa的低分子量蛋白质（LMW）的研究（Torabi等人，2014）。
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