Redox Status-Selective Imaging of Iron in Vegetative and Pathogenic Fungal Cells Using Fluorescent Dyes Synthesized Via Simple Chemical Reactions.

Iron plays a prominent role in various biological processes and is an essential element in almost all organisms, including plant-pathogenic fungi. As a transition element, iron occurs in two redox states, Fe²⁺ and Fe³⁺, the transition between which generates distinct reactive oxygen species (ROS) such as H₂O₂, OH^- anions, and toxic OH· radicals. Thus, the redox status of Fe determines ROS formation in pathogen attack and plant defense and governs the outcome of pathogenic interactions. Therefore, spatially resolved visualization of Fe²⁺ and Fe³⁺ are essential to understand microbial pathogenesis. Here, we report a simple method for synthesis of the redox-state-selective dyes pyrene-tetramethyl piperidinyl oxyl (p-TEMPO) and 4-(4-methylpiperazine-1)-7-nitrobenz-2-oxa-1,3-diazole (MPNBD) for fluorescence microscopy-based imaging of Fe²⁺ and Fe³⁺ ions. Using these dyes, the occurrence and spatial distribution of Fe²⁺ and Fe³⁺ ions in vegetative and pathogenic hyphae of the hemibiotrophic maize anthracnose fungus Colletotrichum graminicola are shown. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.