1. Plant growth conditions
2. Preparation of spore inoculum
NOTE: For details, see Fuchs et al.14.
3. Tissue preparation and fixation
4. Tissue embedding and sectioning
5. Vibratome sectioning
NOTE: The vibratome is equipped with a vibrating razor blade to cut through plant organs/tissue. The vibration speed, amplitude, angle of the blade, and section thickness are all parameters that can be adjusted (see Table of Materials).
6. Specimen clearing
7. Staining procedure
8. Microscopy
With Nile Red that stains lipids and suberin, it is possible to view the pathogen resting spores containing lipids (Figure 3A,B). Hence, using double staining, crisp images can be obtained to look at the pattern of pathogen distribution within the galls. Counterstaining with Calcofluor white creates contrast and helps to simultaneously track xylem development with P. brassicae maturation (Figure 3B).
Xylem formation and development could also be checked by observing autofluorescence in unstained samples (Figure 4A) or by using stains such as Basic Fuchsin that enable fluorescence-based imaging of lignin (Figure 4B).
By using this method, one could track gene expression changes or responses to growth regulators. A perfect example is where Arabidopsis plants harboring pHCA2:erRFP construct were utilized to visualize HIGH CAMBIAL ACTIVITY 2 (HCA2) gene expression in phloem tissue within clubroot galls. HCA2 gene activity has been previously found in meristematicaly active cambium and phloem-lineage cells15. Here, it co-localizes with the phloem in the late stages of P. brassicae-driven gall development, and its activity reflects how P. brassicae increases phloem complexity (Figure 5). The resulting image shows phloem proliferation at the late stage of gall development when the cambium gets fragmented. Figure 5A shows an uncleared hand section of the gall, whereas Figure 5B shows more crisp and localized fluorescent signals obtained by vibratome sectioning followed by tissue clearing. The objects were counterstained with Calcofluor white. Figure 6 compares this image (Figure 6B) with a similar region depicted in resin-embedded and microtome-sectioned galls (Figure 6A) at 21 DPI. Differential cytokinin responses between infected and non-infected plants were assessed by checking the expression of the TCS:GFP (Two Component Signalling) marker16 in developing galls (Figure 7). While imaging weak GFP signals in galls and tissues with secondary thickenings, it is important to note that additional background signal due to autofluorescence of mature xylem cells is also captured during imaging.
Figure 1: Clubroot disease symptoms on oilseed rape (B. napus) and Arabidopsis thaliana (Columbia-0) at 26 DPI with Plasmodiophora brassicae spores. During the course of disease, large galls develop on the entire root system, making it extremely brittle. It concludes by releasing spores into the surrounding soil to promote future infections. The upper parts of the plant body also show signs of poor growth and development. Finally, the infected plants succumb to the devastating effects on growth metabolism and development once the root system gets completely damaged and the plant can no longer cope with the disease. The scale bar represents 1 cm. The -INF stands for mock-inoculated, whereas +INF stands for P. brassicae-inoculated plants. On this occasion, a picture of oilseed rape plants is provided before the soil removal to present healthy root systems. After washing, only the hypocotyl and upper part of the root are collected. Please click here to view a larger version of this figure.
Figure 2: The general workflow. Washed Arabidopsis root systems are (A) dissected, (B) fixed, (C) agarose embedded, (D) mounted, and (E) sectioned on the vibratome. The resulting objects are subjected to tissue clearing (3 days to several weeks at RT in the dark, depending on the tissue type and thickness). (F) Cleared objects can then be stained and inspected under the microscope. (G) Summary of the workflow. Please click here to view a larger version of this figure.
Figure 3: Marking pathogen spores with Nile Red stain. (A,B) Nile Red stains lipids in resting spores, which works perfectly to track P. brassicae maturation. (A) Enlarged cells colonized by P. brassicae and filled with pathogen spores. (B) Mature xylem cells also get stained by Nile Red. The section has been counterstained with Calcofluor white to see the outlay of host cells (A: Objective lens = 20x and section thickness = 60 µm; B: Objective lens = 5x and section thickness = 60 µm). Please click here to view a larger version of this figure.
Figure 4: Tracking the extent of xylem development and maturation. (A) Lignin gives strong autofluorescence upon excitation with UV; therefore, mature xylem can be discriminated relatively easily. (B) Double staining with Basic Fuchsin and Calcofluor gives better results since all cells get stained by the latter dye, while mature xylem is distinctly stained with Basic Fuchsin. In this way, double staining provides images with improved contrast depicting noticeable inhibition of xylogenesis (A: Objective lens = 10x and section thickness = 60 µm; B: Objective lens = 10x and section thickness = 60 µm). Please click here to view a larger version of this figure.
Figure 5: Phloem-specific signal for the HCA2 gene in hypocotyls of P. brassicae-infected plants at 21 DPI. Promoter activity for proHCA2::erRFP harboring transgenic Arabidopsis thaliana can be seen in (A) and (B). Differences can be observed between a non-cleared hand section in panel (A) where the erRFP signal appears diffused due to superimposition and overlapping cell layers, especially in uneven hand sections. On the other hand, (B) shows a vibratome section post-tissue-clearing where the erRFP signal precisely marks the phloem cells in a mature vascular bundle (Objective lens = 20x and section thickness = 60 µm). Please click here to view a larger version of this figure.
Figure 6: Comparison between TB, resin-embedded, and microtome-sectioned galls with the help of fluorescence. (A) A comparison between images of Toluidine Blue (TB), resin-embedded, and microtome-sectioned galls, and (B) a representative object (also presented in Figure 5B) acquired with the help of fluorescence. Xylem cells are labeled with yellow asterisks, the cambial area with vivid green brackets, phloem with cyan asterisks, and Plasmodiophora brassicae-colonized cells with a white PB symbol. Resin-embedded sections (A) provide a good resolution for studying the distribution of resting spores in hypertrophied organs, degree of disease progression, and other processes such as local lignification in resistant plants. However, the protocol described here (B) enables the sensitive observation of gene expression or protein accumulation and the visualization of other physiological changes and important molecules such as lipids (in spores). Please click here to view a larger version of this figure.
Figure 7: Tracking cytokinin signaling responses in hypocotyls of non-infected (-INF) and P. brassicae-infected (+INF) Arabidopsis plants at 16 DPI. TCS::GFP marker was used for characterizing in planta cytokinin responses. Vibratome sections were subjected to clearing treatment followed by staining with Calcofluor white. Based on the image, at 16 DPI, cytokinin responses appear to be largely diminished in infected galls (right panel), while they remain strong, especially in the phloem pool (cells that will eventually differentiate to form phloem tissue), in non-infected plants (left panel) (Objective lens = 5x and thickness = 30 µm). Certain levels of xylem autofluorescence can also be visible. Please click here to view a larger version of this figure.
Clearing Solution (toxic) | ||
Components | Percentage (%) | in 100 mL distilled water |
Xylitol | 10% | 10g |
Sodium Deoxycholate | 15% | 15g |
Urea | 25% | 25g |
10x PBS (Phosphate buffered saline) | 1x PBS (100 mL) | |
NaCl | 8 g | 10 mL 10x PBS + 90 mL distilled water |
KCl | 0.2 g | |
KH2PO4 | 0.24 g | |
Na2HPO4 · 2H2O | 1.81 g | |
distilled water | 100 mL | |
pH | pH adjusted to 7.4 using HCl | |
Autoclave and store at 4 °C. |
Table 1: Composition of the Clearing solution and Phosphate-buffered saline (PBS).
Fluorescent stain/ Tag | Excitation/Emission Wavelengths | Microscope filter set used |
Nile Red | 553/636 nm | filter set 43 |
Xylem Autofluorescence | 380/475 nm | filter set 49 |
Calcofluor White | 405/475 nm | filter set 49 |
Basic Fuchsin | 561/650 nm | filter set 43 |
erRFP | 585/608 nm | filter set 43 |
GFP | 488/509 nm | filter set 38 |
Table 2: Excitation/emission spectra selected for the present study.
2N Sulfuric acid (H2SO4) | Roth | UN2796 | pH adjustment |
Agarose | PRONA | BGQT100 | Embedding |
Basic Fuchsin | BIOSHOP | BSF410.5 | Fluorescent dye |
Calcofluor White | Sigma Aldrich | 18909-100ML-F | Fluorescent dye |
Commercial Bleach | Domestos | ||
Cyanoacrylate/ Instant glue | Kropelka | Adhesive | |
Dimethyl Sulfoxide (DMSO) | BIOSHOP | DMS555.500 | Solvent |
Epifluorescence microscope | Carl Zeiss M2 automated epifluorescence microscope with Colibri LED system | Carl Zeiss M2 | Carl Zeiss Filter Set filter set 38, 43, 49 used |
Fully automated Vibratome | Leica | VT1200 S | |
Lightmeter /Photometer | LI-COR Biosciences | LI-250A + LI-190R quantum sensor | For measuring light intensity within the 400-700nm (PAR) waveband |
Masking tape | For sticking agarose block on mould | ||
Murashige & Skoog Medium (MS Medium) | Duchefa Biochemie | MO222.0050 | Plant Growth Medium |
Nile Red | Sigma Aldrich | N3013-100MG | Fluorescent dye |
Paraformaldehyde PFA | Sigma Aldrich | 158127-100G | Fixative |
Potassium Chloride (KCl) | POCH | 739740114 | PBS component |
Potassium Hydroxide (KOH) | Sigma Aldrich | P1767-250G | pH adjustment |
Potassium Phosphate Monobasic (KH2PO4) | BIOSHOP | PPM302.500 | PBS component |
Sodium chloride (NaCl) | BIOSHOP | SOD001.1 | PBS component |
Sodium Deoxycholate | Sigma Aldrich | D6750-25G | Clearing Solution |
Sodium Phosphate Dibasic (Na2HPO4 · 2H2O) | POCH | 799490116 | PBS component |
Triton X-100 | BIOSHOP | TRX506.100 | Fixative |
Urea | Sigma Aldrich | U5378-100G | Clearing Solution |
Vacuum/Pressure pump and Dessicator | Welch by Gardner Denver | 2522C-02 | For Vacuum Infilteration |
Xylitol | Sigma Aldrich | X3375-25G | Clearing Solution (componenet) |
Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls requires cellular reprogramming and changes in the metabolism of the infected plant. This is necessary to establish a pathogen-oriented physiological sink toward which the host nutrients are redirected. For a complete understanding of this particular plant-pathogen interaction and the mechanisms by which host growth and development are subverted and repatterned, it is essential to track and observe the internal changes accompanying gall formation with cellular resolution. Methods combining fluorescent stains and fluorescent proteins are often employed to study anatomical and physiological responses in plants. Unfortunately, the large size of galls and their low transparency act as major hurdles in performing whole-mount observations under the microscope. Moreover, low transparency limits the employment of fluorescence microscopy to study clubroot disease progression and gall formation. This article presents an optimized method for fixing and clearing galls to facilitate epifluorescence and confocal microscopy for inspecting P. brassicae-infected galls. A tissue-clearing protocol for rapid optical clearing was used followed by vibratome sectioning to detect anatomical changes and localize gene expression with promoter fusions and reporter lines tagged with fluorescent proteins. This method will prove useful for studying cellular and physiological responses in other pathogen-triggered structures in plants, such as nematode-induced syncytia and root knots, as well as leaf galls and deformations caused by insects.
Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls requires cellular reprogramming and changes in the metabolism of the infected plant. This is necessary to establish a pathogen-oriented physiological sink toward which the host nutrients are redirected. For a complete understanding of this particular plant-pathogen interaction and the mechanisms by which host growth and development are subverted and repatterned, it is essential to track and observe the internal changes accompanying gall formation with cellular resolution. Methods combining fluorescent stains and fluorescent proteins are often employed to study anatomical and physiological responses in plants. Unfortunately, the large size of galls and their low transparency act as major hurdles in performing whole-mount observations under the microscope. Moreover, low transparency limits the employment of fluorescence microscopy to study clubroot disease progression and gall formation. This article presents an optimized method for fixing and clearing galls to facilitate epifluorescence and confocal microscopy for inspecting P. brassicae-infected galls. A tissue-clearing protocol for rapid optical clearing was used followed by vibratome sectioning to detect anatomical changes and localize gene expression with promoter fusions and reporter lines tagged with fluorescent proteins. This method will prove useful for studying cellular and physiological responses in other pathogen-triggered structures in plants, such as nematode-induced syncytia and root knots, as well as leaf galls and deformations caused by insects.
Infection of Brassica crops by the soilborne protist Plasmodiophora brassicae leads to gall formation on the underground organs. The formation of galls requires cellular reprogramming and changes in the metabolism of the infected plant. This is necessary to establish a pathogen-oriented physiological sink toward which the host nutrients are redirected. For a complete understanding of this particular plant-pathogen interaction and the mechanisms by which host growth and development are subverted and repatterned, it is essential to track and observe the internal changes accompanying gall formation with cellular resolution. Methods combining fluorescent stains and fluorescent proteins are often employed to study anatomical and physiological responses in plants. Unfortunately, the large size of galls and their low transparency act as major hurdles in performing whole-mount observations under the microscope. Moreover, low transparency limits the employment of fluorescence microscopy to study clubroot disease progression and gall formation. This article presents an optimized method for fixing and clearing galls to facilitate epifluorescence and confocal microscopy for inspecting P. brassicae-infected galls. A tissue-clearing protocol for rapid optical clearing was used followed by vibratome sectioning to detect anatomical changes and localize gene expression with promoter fusions and reporter lines tagged with fluorescent proteins. This method will prove useful for studying cellular and physiological responses in other pathogen-triggered structures in plants, such as nematode-induced syncytia and root knots, as well as leaf galls and deformations caused by insects.