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

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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.

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We thank all participants of the last 3 years of confocal microscopy courses who helped to test this method on various kinds of material, thus helping to improve the method to its present practicable stage; Florian Lemmer and Julian Walther for kindly providing the plant material of S. parviflora and L. abortivum; Friedemann Brauer (Dalhousie University, Halifax, Nova Scotia, Canada) and the anonymous referees for their valuable comments on the manuscript.

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Correspondence to Magnus Rath.

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Figure S3

a and b: Z extraction of the 3D projection in Fig. 2f and g. The high resolution of the initial data set (compare Fig. 2f) allows the analysis of complex structured arbuscules (ar) (S3 a) and their spatial arrangement inside the host plant cell (S3 b). Scale: 100 μm (JPEG 1.26 mb)

Figure S4

a–c: 3D Projection (AMIRA™) of the root fragment of Serapias parviflora (transverse view) with the host plant tissue displayed in grey and the fungus in orange. Tangential view on the initial plane (a) and, after rotation for 180°, last plane (b) of the scan through the root fragment. Although the scan reached 384 μm in depth (see radial view on the projection in c), the quality of the visualization remains high and allows to reconstruct even fine details like hyphae running along the epidermal surface (see arrowhead in b) or, if the fungal channel is displayed alone, hyphal coils forming a colonisation cluster inside the root cortex parenchyma (d). Scale: a, b, d=150 μm; c=384 μm (JPEG 3.33 mb)

Figure S5

3D projection (AMIRA™) of the root fragment of Polygala rupestris non-specifically stained with acid fuchsine. Fungal structures like degenerated arbuscules (da) and fungal hyphae (hy) are well distinguishable from host plant tissue cells. Scale: 100 μm (JPEG 1.08 mb)

Tomographic animation (AMIRA™) of the CLSM scan of a Polygala paniculata root in Fig. 2f (MPG 31821 kb)

Tomographic animation (AMIRA™) of the CLSM scan of a Triticum aestivum leaf infested by Zymoseptoria tritici (compare Fig. 2e) (MPG 71515 kb)

Animation S6

Tomographic animation (AMIRA™) of the CLSM scan of a Polygala paniculata root in Fig. 2f (MPG 31821 kb)

Animation S7

Tomographic animation (AMIRA™) of the CLSM scan of a Triticum aestivum leaf infested by Zymoseptoria tritici (compare Fig. 2e) (MPG 71515 kb)

Table 1

Collecting data of the plant material used in this study (DOC 25.5 kb)

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Rath, M., Grolig, F., Haueisen, J. et al. Combining microtomy and confocal laser scanning microscopy for structural analyses of plant–fungus associations. Mycorrhiza 24, 293–300 (2014).

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  • Confocal laser scanning microscopy
  • Microtomy
  • Three-dimensional structural analysis
  • Mycorrhiza