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

光控制着丝状真菌的许多重要过程，如繁殖和致病性。真菌天生具有几个光传感器，它们分别对大量的波长做出反应，最大的波长分别是蓝色、绿色或红色。绿光感应是由真菌紫红质进行的，属于微生物紫红质，近年来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7个跨膜螺旋组成，形成一个内部口袋，用于全视网膜发色团，该发色团通过质子化的希夫碱基与蛋白质共价结合。虽然已知真菌视紫红质在光激活时作为质子泵或感觉蛋白，但对其生理功能和生物学作用的详细了解仍然缺乏。在本项目中，我们将分析筛选出的真菌紫红质的分子功能和生物学作用。我们将结合多种生物物理方法来表征来自丝状真菌粗神经孢子菌（Nop-1）、藤黑镰刀菌（CarO, OpsA）、斑点褐球菌（PhaeoRD1, PheoRD2）、斑点细球菌（LR）和麦氏黑穗病菌（UmOps1-3）的红紫红质分子功能。我们将使用膜片钳技术研究在哺乳动物细胞或酵母中异种表达的真菌视紫红质的泵功能，重点研究转运离子种类、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紫红质，并利用闪光分解光谱分析光循环的中间体。为了深入了解真菌视紫红质的生物学作用，我们将荧光视紫红质在N. crassa， F. fujikuroi和U. maydis的菌丝中表达。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。我们将使用ph敏感染料在活的fujikuroi菌丝中观察真菌视紫红质活化后局部ph的变化。此外，为了弄清真菌紫红质的生物学功能，我们将比较几种F. fujikuroi菌株（紫质缺失突变体和紫质缺失突变体以及几种光受体）在不同光照条件（波长/强度）和不同营养供应下的生长参数。由于气候变暖和集约化农业，真菌感染正在增加。因此，迫切需要获得真菌对环境，特别是光的反应的详细知识。从长远来看，该项目的结果可能支持开发急需的杀菌剂和/或生物技术方法中真菌生长的增强。

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.