Abstract
Single-molecule localization microscopy has boosted our understanding of biological samples by offering access to subdiffraction resolution using fluorescence microscopy methods. While in standard mammalian cells this approach has found wide-spread use, its application to filamentous fungi has been scarce. This is mainly due to experimental challenges that lead to high amounts of background signal because of ample autofluorescence. Here, we report the optimization of labeling, imaging and data analysis protocols to yield the first single-molecule localization microscopy images of the filamentous fungus Trichoderma atroviride. As an example, we show the spatial distribution of the Sur7 tetraspanin-family protein Sfp2 required for hyphal growth and cell wall stability in this mycoparasitic fungus.
Highlights
In recent years, more and more problems have arisen due to the excessive use of chemical fungicides for plant pest control
We adapted their procedure for the filamentous fungus T. atroviride
To allow for single-molecule localization microscopy (SMLM), we stained the cells with a red fluorescent AF647-conjugated anti-GFP nanobody recognizing the expressed GFP fusion construct (Figure 1A)
Summary
More and more problems have arisen due to the excessive use of chemical fungicides for plant pest control. One possible solution makes use of the mycoparasitic behavior of certain fungi to protect plants from phytopathogen attack. Fungal mycoparasitic behavior has been known for decades, we are only beginning to understand the underlying molecular interactions and spatial protein organizations in mycoparasitic fungi. In the last few years, superresolution optical microscopy has enabled the analysis of the spatial distribution of biomolecules in cells at length scales of a few tens of nanometers [2]. Single-molecule localization microscopy (SMLM) utilizes high precision to localize single-molecule signals in order to construct a localization map of the dye molecules bound to the biomolecule of interest. The literature on SMLM applied to study protein organization in filamentous fungi is scarce [3,4], likely due to experimental challenges imposed by the fungus compared to animal
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