Interdisciplinary Analysis of Fungal Rhodopsins and Their Physiological Function in Mycelia.

真菌视紫红质及其在菌丝体中的生理功能的跨学科分析。

• 批准号: 251151058
• 负责人: Dr. Ulrich Terpitz
• 金额: --
• 依托单位: Lehrstuhl für Biotechnologie und Biophysik
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2017-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/251151058?language=en
• 关键词: Interdisciplinary; Analysis; Fungal; Rhodopsins; Their

Light is controlling many substantial processes in filamentous fungi such as reproduction and pathogenicity. Fungi naturally possess several light sensors which react to a broad plethora of wavelengths with maxima in the blue, green or red, respectively. Green light sensing is performed by the fungal rhodopsins belonging to the the microbial rhodopsins, which recently became famous for their use in optogenetics. Fungal rhodopsins are widespread in the fungal kingdom and consist of seven transmembrane helices forming an interior pocket for the chromophore all-transretinal, which is covalently bound to the protein via a protonated Schiff-base. Though it is known that upon light-activation fungal rhodopsins act as proton pumps or sensory proteins, detailed knowledge of their physiological function and biological role is still missing. In this project we will analyze the molecular function and the biological role of elected fungal rhodopsins. We will combine a number of biophysical methods to characterise the molecular function of rhodopsins from the filamentous fungi Neurospora crassa (Nop-1), Fusarium fujikuroi (CarO, OpsA), Phaeosphaeria nodorum (PhaeoRD1, PheoRD2), Leptosphaeria maculans (LR), and Ustilago maydis (UmOps1-3). We will use Patch-clamp techniques to investigate the pump-function of fungal rhodopsins heterologously expressed in mammalian cells or yeasts focusing on transported ion-species, kinetics, voltage dependency, turnover rate, and applicability in optogenetical approaches. In addition we will purify elected fungal rhodopsins and analyse intermediates of the photocycle by flash-fotolysis spectroscopy. In order to get principle insights into the biological role of fungal rhodopsins we will express fluorescent rhodopsins in hyphae of N. crassa, F. fujikuroi, and U. maydis. We will investigate fixed, immunostained mycelia by confocal laser scanning microscopy (cLSM) and super-resolution localisation microscopy (PALM / dSTORM). We will use pH-sensitive dyes in living F. fujikuroi hyphae to observe local pH-changes upon fungal rhodopsin activation. Moreover, in order to figure out, what could be the biological function of the fungal rhodopsins, we will compare the growth parameters of several F. fujikuroi strains (WT and deletion mutants of rhodopsins and several light receptors) cultivated under varying light conditions (wavelength/intensity) and different nutrient supply.Fungal infections are increasing due to climate warming and intensive agriculture. Thus, it is of urgent need to gain detailed knowledge of fungal response to their environment, especially light. Results of this project may in the long-term perspective support the development of urgently needed fungicides and/or the enhancement of fungal growth in biotechnological approaches.

光控制着丝状真菌的许多重要过程，如繁殖和致病性。真菌天然地具有几种光传感器，其分别对蓝色、绿色或红色中具有最大值的大量波长作出反应。绿色光传感是由真菌视紫红质进行的，该视紫红质属于微生物视紫红质，最近因其在光遗传学中的应用而闻名。真菌视紫红质广泛存在于真菌界，由七个跨膜螺旋组成，形成发色团全transretinal的内部口袋，其通过质子化的席夫碱与蛋白质共价结合。虽然已知真菌视紫红质在光激活时充当质子泵或感觉蛋白，但对其生理功能和生物学作用的详细知识仍然缺失。本研究将分析选出的真菌视紫红质的分子功能和生物学作用。我们将结合联合收割机多种生物物理学方法，对丝状真菌粗糙脉孢菌（Neurospora crassa）（Nop-1）、藤仓镰刀菌（Fusarium fujikuroi）（CarO，OpsA）、颖枯褐球腔菌（PhaeoRD 1，PheoRD 2）、斑点细球腔菌（Leptosphaeria maculans）（LR）和玉米黑粉菌（UmOps 1 -3）的视紫红质的分子功能进行研究。我们将使用膜片钳技术研究在哺乳动物细胞或酵母中异源表达的真菌视紫红质的泵功能，重点是转运离子种类、动力学、电压依赖性、周转率和光遗传学方法的适用性。此外，我们将纯化选定的真菌视紫红质和分析中间体的光循环的闪光fotolysis光谱。为了深入研究真菌视紫红质的生物学功能，我们将在N. crassa，F. fujikuroi和U.玉米粉我们将通过共聚焦激光扫描显微镜（cLSM）和超分辨率定位显微镜（PALM / dSTORM）研究固定的免疫染色菌丝体。我们将在活体F. Fujikuroi菌丝，以观察真菌视紫红质活化后的局部pH变化。此外，为了弄清楚真菌视紫红质的生物学功能，我们将比较几种真菌的生长参数。Fujikuroi菌株（视紫红质和几种光受体的WT和缺失突变体）在不同的光条件（波长/强度）和不同的营养供应下培养。因此，迫切需要详细了解真菌对其环境（尤其是光线）的反应。从长远来看，该项目的结果可能会支持开发急需的杀真菌剂和/或在生物技术方法中增强真菌生长。