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

光控制着丝状真菌的许多实质性过程，如繁殖和致病。真菌天生就有几个光传感器，它们对多种波长都有反应，最大值分别是蓝色、绿色或红色。绿光传感是由真菌视紫红质执行的，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中，它由7个跨膜螺旋组成，形成发色团的内袋，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后作为质子泵或感觉蛋白发挥作用，但对其生理功能和生物学作用仍缺乏详细的了解。在这个项目中，我们将分析选择的真菌视紫红质的分子功能和生物学作用。我们将结合一些生物物理方法来表征丝状真菌粗面脉孢霉(NOP-1)、藤红镰刀菌(CARO，OPSA)、诺多氏褐孢杆菌(PhaeoRd1，PheoRD2)、斑点纤毛菌(Leptosphaeria maculans，LR)和玉米黑粉菌(Ustilago maydis，UmOps1-3)的视紫红蛋白的分子功能。我们将使用膜片钳技术来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，重点是转运的离子种类、动力学、电压依赖性、周转率和光遗传学方法的适用性。此外，我们还将提纯选定的真菌视紫红质，并通过闪光分解光谱分析光循环的中间产物。为了深入了解真菌视紫红质的生物学作用，我们将在粗毛拟青霉、富士黑曲霉和玉米赤霉菌的菌丝中表达荧光视紫红素。我们将用共聚焦激光扫描显微镜(CLSM)和超分辨定位显微镜(Palm/dSTORM)研究固定的、免疫染色的菌丝体。我们将在活菌丝中使用pH敏感的染料来观察真菌视紫红质激活时局部pH的变化。此外，为了弄清真菌视紫红质的生物学功能，我们将比较几个藤库氏菌株(视紫红质WT和缺失突变体以及几个光受体)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，迫切需要详细了解真菌对其环境，特别是光照的反应。从长远来看，这一项目的结果可能有助于开发急需的杀菌剂和/或促进生物技术方法中的真菌生长。

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.