Frozen in time: a new method using cryo-scanning electron microscopy to visualize root-fungal interactions

Area of Science:

• Plant Pathology
• Microscopy

Background:

• Wheat (Triticum aestivum) is susceptible to Rhizoctonia solani AG-8, a pathogenic fungus causing significant crop losses.
• Conventional sample preparation methods for microscopy can introduce artifacts, hindering accurate visualization of fungal-plant interactions.

Purpose of the Study:

• To develop and validate a new sample preparation method for cryo-scanning electron microscopy (cryo-SEM).
• To visualize the three-dimensional, in situ internal infection of wheat roots by Rhizoctonia solani AG-8 without artifacts.

Main Methods:

• A novel cryo-SEM sample preparation technique involving rapid freezing in liquid nitrogen.
• Cryo-planing and etching (sublimation) of frozen samples at -80°C for a critical duration (up to 9 minutes) within the microscope column.
• In situ observation of preserved plant and fungal structures.

Main Results:

• The new method successfully preserved fungal hyphae and wheat root cells in situ, avoiding distortions from conventional preparation.
• Root and fungal structures, including cell walls and hyphal architecture, were clearly visualized in three dimensions, irrespective of infection severity.
• The method allowed detailed observation of hyphal invasion within and between root cells.

Conclusions:

• This rapid cryo-SEM method provides high-fidelity, three-dimensional in situ visualization of fungal invasion in plant tissues.
• The technique has broad applicability for studying diseases caused by necrotrophic fungi in various plant-pathogen systems.