Our protocol is a simplified method that allows researchers to visualize early infection steps of wild type and genetic fungal mutants without the use of expensive equipment. The main advantage of this technique is the use of basic lab supplies to answer complex biological questions and for the rapid screening of fungal mutants for further analysis. A difficult portion of this technique is the treatment of the sheath.
The sheath is a thin layer of cells that can be easily damaged, so careful handling is essential for a successful experiment. To begin, select the barley grown until the second leaf stage. And using sterile scissors, cut the barley plant just above the soil line.
Using forceps and a scalpel, carefully cut the sheath of the first leaf that is open longitudinally. And using the forceps, remove it from the base of the second leaf. Using a scalpel, cut most of the first leaf away from the sheath, leaving only 0.5 inches of the leaf tissue for mounting.
Place the first leaf flat in a sterile 60 millimeter Petri dish containing a wet paper towel to maintain humidity inside the plate. Tape the leaf tissue to the bottom of the Petri dish. Select a nine to 12-day-old Magnaporthe oryzae culture plate and add 0.5 to two milliliters of sterile water to it.
Using a sterile inoculation loop, gently scrape the mycelia to release the attached conidia. Carefully pipette the conidial suspension into a microcentrifuge tube containing a small piece of cheese cloth to filter out any large pieces of mycelium from the conidial suspension. If necessary, dilute the spore concentration with sterile water to five times 10 to the fourth spores per milliliter, as too high of a concentration makes imaging individual infection sites challenging.
Depending on the sheath size, carefully pipette 25 to 50 microliters of the conidial suspension inside the rolled leaf sheath. Next, fill four or five 500 milliliter beakers with double distilled water and heat until steaming. Hold the lid of the Petri dish over one of the steaming beakers to trap humidity inside the plate.
Stack the infected leaf sheath plates and surround them with the remaining hot beakers, as it creates a humid, moist environment for the spores to germinate. Protect this setup from light by covering it with a solid colored rubber or plastic box and leaving it undisturbed for 48 hours or the desired time point for imaging. Prepare the stain by mixing freshly diluted 45%acetic acid and 0.1%trypan blue.
Aliquot one milliliter of the dye solution into microcentrifuge tubes. Using a scalpel, carefully cut the leaf sheath away from the tape. Using forceps, place the sheath into the microcentrifuge tube and ensure it is completely submerged in the dye solution.
Allow the tubes to stand for two hours in a 40 degree Celsius heat block or water bath for the dye to penetrate the leaf. Next, carefully rinse the leaf sheaths three times in 60%fresh glycerol to remove the extra dye. Keep the sheath in glycerol until ready to mount on slides.
Place the sheath on a clean glass slide and add a few drops of 60%glycerol. Using a dissecting microscope and two pairs of forceps, carefully unroll the sheath, leaving the inoculated center facing up. Holding the sheath open with the forceps, place the cover slip on top to prevent the sheath from curling and blocking the infection site.
Seal the cover slip using nail polish for long-term storage or tape short-term storage. Observe the slides under a compound light microscope. Take basic images with a smartphone by mounting a cell phone adapter to the microscope.
For Android devices, adjust the camera application settings to flash off, disable top shot, disable automatic adjustment of brightness and shadows, and set the photo resolution to full. After mounting the cell phone, take an image of a scale micrometer with a desired objective for acquiring the data. Adjust the phone's zoom to 2.5 x and keep it consistent to maintain a consistent pixel size.
The center of the sheath houses the largest concentration of spores and infecting appressoria. Therefore, aim for nine to 12 images of each sheath to obtain significant numbers for statistical analysis. The infection sites of the sheath were imaged using a smartphone and smartphone microscope adapter after staining with trypan blue.
The heat and acetic acid in the staining procedure softens the leaf tissue gently. The post-staining rinses in 60%glycerol not only remove the excess stain but help reduce the light scattering caused by the leaf and improve the image quality. Deviations from the staining protocol can result in suboptimal results, as depicted here.
Barley infected with 4091 wild type produced successfully infected cells identified by the presence of infected hyphae inside the leaf sheath tissue. In the case of the mutant J99A infection, successful development of appressoria and penetration pegs was noticed but did not produce invasive hyphae. Timing's important for infection-based assays.
Appropriately aged plants and fungal spores are essential for success. In this study, we looked at the presence or absence of the infection structures. Additional experiments could answer questions regarding the phenotype of the infection structure and that of the invading hyphae.
Our methodology is not new, but a simplified, accessible version of a more complex, expensive experiment.
