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

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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.