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

The present protocol describes an optimized method for the histological observation of galls induced by Plasmodiophora brassicae. Vibratome sections of hypocotyls are cleared before fluorescence imaging to study the involvement of transcription factors and phytohormones during disease progression. This protocol overcomes resin embedding limitations, enabling in planta visualization of fluorescent proteins.

Plasmodiophora brassicae is the soil-borne obligatory biotrophic pathogen that attacks plants from the Brassicaceae family. Upon infection, pathogen triggers the development of galls on the underground parts of the plant, hence the name of the disease clubroot. The development of galls is accompanied by very complex reprogramming.

Therefore, the possibility to track internal changes during disease is crucial for understanding this plant-pathogen interaction. Our protocol improves imaging of complex, thick, and galls. It eases visualization of changes in gene expression, physiological responses, phytohormone balance, disease progression, spore distribution, and local lignification.

The protocol preserves fluorescent proteins within samples, allowing storage and processing of objects over a long time. It also enables the tracking of biochemical and structural changes accompanying disease progression. Demonstrating the procedure will be Sara Blicharz, post-doctoral researcher in our group, Deeksha Singh, PhD student from our laboratory, and Kornel Michalak, PhD student from Professor Agnieszka Bagniewska-Zadworna group at Adam Mickiewicz University.

Begin by preparing the plant tissue. Carefully remove soil from the root systems of clubroot-infected and non-infected plants. Clean thoroughly with water and collect hypocotyls and galls into microcentrifuge tubes.

Fix the samples in 200 to 500 microliters of paraformaldehyde fixative for one hour at room temperature by applying a constant vacuum of 700 millibars using a vacuum pump. Ensure that the object is completely immersed in the paraformaldehyde solution. Use 4%agarose for embedding non-infected hypocotyls and infected galls.

Boil the solution to dissolve agarose and pour it when it cools down while it is still viscous. Dry the object slightly on a tissue for a few seconds to remove excess paraformaldehyde. Use toothpicks or forceps to embed and orient the plant object in agarose carefully.

To prevent oblique sections, ensure that the object is oriented and molded correctly so that its axis is perpendicular to the plane of the vibratome blade. To accelerate agarose solidifying, place a petri or multiculture plate at four degrees Celsius for 10 minutes. Using a blade, carefully cut and mold the object within an agarose block, noting the preferred orientation for sectioning.

Stick the agarose block or large gall to properly mount it on the specimen holder using cyanoacrylate or instant glue and masking tape. Adjust the thickness for sections, speed, and vibration amplitude according to the gall's thickness and size. Add distilled water to the water bath and begin with sectioning.

Carefully collect sections using forceps or brush and transfer them into a microcentrifuge tube containing one milliliter of 1X PBS buffer. Remove 1X PBS from the microcentrifuge tube and add 200 to 500 microliters of clearing solution. Replace the clearing solution with the new one if its color changes after some time during sample processing.

Perform calcofluor-white staining for the cell walls of the outlaying plant cells by preparing 5%of calcofluor-white stain in the clearing solution. Then stain the cells in the dark for at least five minutes. Stain lipids with Nile red and lignins with Basic Fuchsin lignin staining as described in the text manuscript and mount the cleared sections on the microscopy slide.

Then observe the cells under an epifluorescence or a confocal microscope. Use multiple acquisition modes for imaging more than one fluorescence spectrum simultaneously. Disease dynamics and pathogen accumulation was tracked in galls colonized by Plasmodiophora brassicae, and large cells with P.brassicae resting spores were observed after Nile red staining.

Changes in xylem differentiation upon P.brassicae infection was visualized using Basic Fuchsin staining. The Arabidopsis plants harboring pHCA2:erRFP construct were utilized to visualize HCA2 gene expression in phloem tissue within clubroot galls. HCA2 co-localized with the phloem in the late stages of P.brassicae-driven gall development, and the gene activity reflected the mechanism by which P.brassicae increases phloem complexity.

At 16 days post-inoculation, differential cytokinin responses between infected and non-infected plants were assessed by checking the expression of the TCS:GFP marker in developing galls. The cytokinin responses appear to be significantly diminished in infected galls, while they remained strong, especially in the phloem pool, in non-infected plants. Most critical steps are fixation in PFA, embedding, and sectioning.

For vibratome sectioning, one needs to optimize and carefully adjust speed, amplitude, and thickness parameters to obtain good-quality sections. Vibratome sectioning and tissue clearing techniques have paved the way for enhancing microscopic analysis of physiological responses during plant-microbe interactions and tissues exhibiting secondary growth with complex cellular anatomy.