Combining microtomy and confocal laser scanning microscopy for structural analyses of plant–fungus associations

Abstract

The serious problem of extended tissue thickness in the analysis of plant-fungus associations was overcome using a new method that combines physical and optical sectioning of the resin-embedded sample by microtomy and confocal microscopy. Improved tissue infiltration of the fungal-specific, high molecular weight fluorescent probe wheat germ agglutinin conjugated to Alexa Fluor® 633 resulted in high fungus-specific fluorescence even in deeper tissue sections. If autofluorescence was insufficient, additional counterstaining with Calcofluor White M2R or propidium iodide was applied in order to visualise the host plant tissues. Alternatively, the non-specific fluorochrome acid fuchsine was used for rapid staining of both, the plant and the fungal cells. The intricate spatial arrangements of the plant and fungal cells were preserved by immobilization in the hydrophilic resin Unicryl™. Microtomy was used to section the resin-embedded roots or leaves until the desired plane was reached. The data sets generated by confocal laser scanning microscopy of the remaining resin stubs allowed the precise spatial reconstruction of complex structures in the plant-fungus associations of interest. This approach was successfully tested on tissues from ectomycorrhiza (Betula pendula), arbuscular mycorrhiza (Galium aparine; Polygala paniculata, Polygala rupestris), ericoid mycorrhiza (Calluna vulgaris), orchid mycorrhiza (Limodorum abortivum, Serapias parviflora) and on one leaf-fungus association (Zymoseptoria tritici on Triticum aestivum). The method provides an efficient visualisation protocol applicable with a wide range of plant-fungus symbioses.

The transcription factor Zt107320 affects the dimorphic switch, growth and virulence of the fungal wheat pathogen Zymoseptoria tritici

“…Morphology and development of Z. tritici within and on the surface of leaves of B. distachyon inbred line Bd21 were analysed by confocal laser‐scanning microscopy (CLSM) as described previously (Haueisen et al , ). Analyses of compatible Z. tritici infections on T. aestivum 'Obelisk' were conducted by combining microtomy and CLSM as previously described (Rath et al , ). Distinct areas of the second leaf of 12‐day‐old (Bd21) and 14‐day‐old (wheat) seedlings were brush‐inoculated with 1 × 10 7 cells/mL in 0.1% Tween 20.…”

Semiautomated confocal imaging of fungal pathogenesis on plants: Microscopic analysis of macroscopic specimens

“…Since it is also widely used to stain the plasma membrane, it was quite unexpected that it did not stain the plant tissue. Interestingly, WGA has been used previously to detect fungi in resin embedded and sectioned samples using standard electron microscopy protocols (Meyberg, 1988; Rath et al, 2013). Another significant advantage of using WGA over other dyes is that it can be conjugated to dyes with varying fluorescent properties.…”

Laser ablation tomography (LATscan) as a new tool for anatomical studies of woody plants

“…Because LATscan enabled direct visualization of the spatial variation of some (lignin and suber) cell wall components without any chemical treatment or staining of cell walls, it can be considered another tool to facilitate the integration of histology and chemical analysis of cell walls in plants. In addition to gross anatomy, this approach may be particularly useful for studying complex systems such as wounding experiments (e.g., grafting), plant-pathogen associations or parasitic plants-host interactions, which normally requires complementary chemical analysis (e.g., histochemistry, fluorescence microscopy) to identify tissues that are specific to the pathogen/parasitic plant or host plant, or to differentiate chemical compounds deposited in boundary layers of wounded plants (Rittinger et al ., 1987; Rath et al ., 2014; Navarro et al ., 2019; Pellissari et al ., 2022).…”