Opsin 1 and Opsin 2 of the Corn Smut Fungus Ustilago maydis Are Green Light-Driven Proton Pumps

Abstract

In fungi, green light is absorbed by rhodopsins, opsin proteins carrying a retinal molecule as chromophore. The basidiomycete Ustilago maydis , a fungal pathogen that infects corn plants, encodes three putative photoactive opsins, called ops1 (UMAG_02629), ops2 (UMAG_00371), and ops3 (UMAG_04125). UmOps1 and UmOps2 are expressed during the whole life cycle, in axenic cultures as well as in planta , whereas UmOps3 was recently shown to be absent in axenic cultures but highly expressed during plant infection. Here we show that expression of UmOps1 and UmOps2 is induced by blue light under control of white collar 1 (Wco1). UmOps1 is mainly localized in the plasma membrane, both when expressed in HEK cells and U. maydis sporidia. In contrast, UmOps2 was mostly found intracellularly in the membranes of vacuoles. Patch-clamp studies demonstrated that both rhodopsins are green light-driven outward rectifying proton pumps. UmOps1 revealed an extraordinary pH dependency with increased activity in more acidic environment. Also, UmOps1 showed a pronounced, concentration-dependent enhancement of pump current caused by weak organic acids (WOAs), especially by acetic acid and indole-3-acetic acid (IAA). In contrast, UmOps2 showed the typical behavior of light-driven, outwardly directed proton pumps, whereas UmOps3 did not exhibit any electrogenity. With this work, insights were gained into the localization and molecular function of two U. maydis rhodopsins, paving the way for further studies on the biological role of these rhodopsins in the life cycle of U. maydis .

Expansion Microscopy for Cell Biology Analysis in Fungi

“…The U. maydis strains expressing opsin1 fused to enhanced green fluorescent protein (eGFP) were described in detail before (Panzer et al, 2019) and derived from the wild type isolate FB1 (Banuett and Herskowitz, 1989). Either the strain FB1 UmOps1 P crg ::UmOps1-eGFP K1 or FB1 P crg ::UmOps1-eGFP KA was used for ExM experiments.…”

“…Either the strain FB1 UmOps1 P crg ::UmOps1-eGFP K1 or FB1 P crg ::UmOps1-eGFP KA was used for ExM experiments. U. maydis sporidia were grown as described before (Panzer et al, 2019). Sporidia were grown in PDB for 15-24 h at 28 • C and 100 rpm, harvested by centrifugation (4000 × g, 3 min), washed once in ddH 2 O and resuspended in ddH 2 O to a final density of 6.5 x 10 5 sporidia/mL.…”

“…Isotropically expanded fungal cells (∼4.5-fold) were submitted to CLSM and SIM (Gustafsson et al, 2008) for superresolution fluorescence imaging. We imaged Ustilago maydis sporidia expressing a fluorescent version of the membrane rhodopsin UmOps1 (Panzer et al, 2019) as well as Fusarium oxysporum hyphae expressing histone H1-mCherry (Ruiz-Roldan et al, 2010) and the F-actin marker Lifeact-sGFP (Fernández-Ábalos et al, 1998;Riedl et al, 2008). Moreover, we show that the ExM protocol can be used successfully for superresolution fluorescence imaging of the clinically relevant human pathogen Aspergillus fumigatus.…”

Ustilago maydisserves as a novel production host for the synthesis of plant and fungal sesquiterpenoids

“…The same strategy was applied for the identification of enzymes of the prenyl phosphate module, producing geranylgeranyl-diphosphate via chain elongation from IPP and DMAPP (GGPP; Figure 1A, Table 1). The carotenoid module has been predicted before and its main function is the production of retinal that serves as chromophore for photoactive opsin channels Ops1-3 (Estrada et al, 2009;Panzer et al, 2019). The cleavage reaction of -carotene into two retinal molecules is catalysed by Cco1 (-carotene cleavage oxygenase; Estrada et al, 2009).…”

“…It was already shown that deletion of cco1 resulted in the accumulation of -carotene (Estrada et al, 2009). Loss of retinal synthesis causes no mutant phenotype under standard growth conditions or during pathogenic development, thus the biological function of opsins in U. maydis is as yet unclear (Estrada et al, 2009;Panzer et al, 2019).…”