Visualization of the protocol as a video recording of the procedures

Introduction to the technique for isolating epiphytic, epidermal, and fungal haustoria from barley leaves infected with Blumeria graminis powdery mildew. The video explains the challenges of working with obligate biotrophic fungi and outlines the overall workflow.

Set up the work surface with necessary tools and prepare the cellulose acetate solution in a 15ml tube. Depending on the intended analysis, prepare MES buffer float and liquid nitrogen-filled falcon tubes for snap-freezing.

Cut individual barley plants at the sheath and immerse primary leaves in cellulose acetate solution five times. Allow acetone to evaporate for five to eight minutes until the epiphytic mycelia-containing layer hardens and begins to peel away.

Remove prepared leaves from pasteur pipettes and carefully peel off the epiphytic mycelia layer using a razor blade to create an initial cut. Extract and peel the abaxial epidermis by gripping and pulling in smooth motions while maintaining resistance.

Mix dissected epidermal strips with cellulase enzyme (Onazuka R10) in MES buffer and incubate at 28 degrees Celsius for 2 hours with gentle shaking. Cellulase enzymatically breaks down epidermal cell walls and releases haustoria into the buffer.

Filter the enzyme-treated suspension through a 70 µm nylon mesh sieve to separate released haustoria from epidermal debris. Centrifuge the filtered liquid at 3270 G for 10 minutes at 4 degrees Celsius to pellet isolated haustoria.

Re-suspend the haustoria pellet in PBS buffer and transfer to an Eppendorf tube. Add alexaflor 488 wheat germ agglutinin to a final concentration of 10 µM per milliliter and mix by inversion.

Load the stained haustoria suspension onto a microscope slide and cover with a cover glass. Use epifluorescence microscopy at multiple magnifications to visualize the stained haustoria, identifying intact structures and distinguishing them from cellular debris and chloroplast impurities.