Combining Clearing and Fluorescence Microscopy for Visualising Changes in Gene Expression and Physiological Responses to <em>Plasmodiophora brassicae</em>

Abstract

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.

MYB68 orchestrates cork differentiation by regulating stem cell proliferation and suberin deposition

“…For gene expression analysis, we employed two approaches: vibratome cross-sections and whole-mounts. First, in the case of vibratome cross-sections, 1 cm-root samples (0.5 cm below the root junction) were collected in water and then fixed using a 4% PFA solution (in 1xPBS) following the protocol described in (Singh et al ., 2022; Ursache et al ., 2018). The fixation was performed under vacuum for 1-2 hours, and then the root parts were rinsed twice for 1 minute in 1xPBS and embedded in 6% agarose blocks.…”

“…Approximately 20 vibratome cross-sections (50-100µm) were collected in water and then transferred to a clearing solution (ClearSee (Ursache et al ., 2018)). Staining of vibratome cross-sections was prepared following the protocol described in (Singh et al ., 2022; Ursache et al ., 2018). Briefly, for Calcoflour White staining, vibratome cross-sections were stained with a 0.05% Calcoflour White solution (in ClearSee) for 5 minutes, while for Direct Red staining, vibratome sections were incubated for 30 minutes in a 0.05% Direct Red solution (in ClearSee).…”

Imaging plant cell walls using fluorescent stains: The beauty is in the details

“…14 For instance, a combination of Nile Red and CW was used to visualize the Plasmodiophora brassicae-caused gall formation in A. thaliana roots. 136 Auramine O is another, less specific fluorescent stain that has been proven to be suitable to visualize suberin and cutin in plants. In addition, it strongly stains lignin and, when combined with lignin stains of different spectra, such as BF, could potentially serve as a dual-labelling system for imaging lignin-suberin and lignin-cutin pairs.…”