Detached Maize Sheaths for Live-Cell Imaging of Infection by Fungal Foliar Maize Pathogens

So our team aims to understand fungal pathogenicity mechanisms in plants to improve disease management. We are currently exploring how the maize autochthonous fungus regulates protein secretion beyond its housekeeping function. Our protocol shows high synchronicity across replications and reproducibility of fungal colonization.

Also, with our assays, there is no need for clearing or fixating samples before imaging them in real-time. The method offers a variety of applications in plant-pathogen interactions and contributed to detailed studies on the mechanisms of fungal pathogenicity in maize and comparative genomic analysis. To begin, use heavy duty scissors or shears to cut off the cap and 0.5 millimeters from the conical bottom of a 1.5 milliliter microcentrifuge tube.

Place a piece of glass wool in the cut tube to cover the center hole. Place one assembled glass wool filter unit into a sterile 1.5 milliliter microcentrifuge tube and label the tube. Next, cut a non-skirted 96-well PCR plate into six two-column support racks.

Flip the two-column racks so the conical bottoms of the wells face upwards. Place the rack in a glass petri dish containing moist filter paper and cover it with the lid to create a humidity chamber for the leaf sheaths. To harvest conidia from the sporulating colletotrichum graminicola, add three to four milliliters of sterile deionized water into the plate.

Using a sterile conical tip pestle, scrape evenly across the entire plate to loosen the spores from the agar. Aseptically, add one milliliter of spore suspension onto a glass wool filter unit and allow the spores to flow into the collection tube by gravity. Centrifuge the filtered spore suspension at 3, 500G for five minutes and pour off the liquid into an autoclavable container.

Add one milliliter of sterile deionized water into the collection tube and gently agitate to resuspend the pelleted spores. Again, centrifuge the suspension and resuspend the spores for quantification in 300 to 500 microliters of deionized water. Use a hemocytometer under a compound microscope at hundred times magnification to determine the spore concentration.

Then prepare 500, 000 spores per milliliter suspension with deionized water. To remove the sheath from the first true leaf of week two stage seedlings, run a thumbnail along the overlapping margin of the sheath and gently loosen it from the shoot. Cut the recovered leaf sheath into three to five centimeter segments.

Carefully unroll each segment to expose the inner epidermal layer. Apply 20 microliters of fungal spore suspension on the inner surface at the center of the sheath above the mid rib. Place the inoculated leaf sheaths horizontally with a mid rib positioned at the bottom in a glass petri plate containing moistened filter paper.

Next, place the small humidity chambers into a clear storage box lined with moistened germination paper. Cover the storage box with a lid and incubate at 23 degrees Celsius under continuous illumination for the intended time course. Regularly check the boxes for signs of disease on the sheaths.

To begin, gently rinse the maize leaf sheaths inoculated with fungal spores in deionized water. Place the sheath on a clean glass microscope slide with the adaxial surface facing down and the abaxial surface uppermost. Using a single edge razor blade, trim approximately one centimeter off the sheath ends and remove most lamina tissue on either side of the mid rib.

Hold the razor blade at a 90 degree angle to shave tissue from the abaxial mid rib, exposing the epidermal layer on the adaxial surface above the inoculated spot. Carefully lift the sheath section from one edge and transfer it onto a new slide with the adaxial surface facing the uppermost, closest to the objective lens. Apply 60 microliters of deionized water to the section and place a glass cover slip without introducing air bubbles.

Stenocarpella maydis rapidly invaded epidermal cells directly via undifferentiated hyphal swellings. While colletotrichum graminicola produced melanized appressoria. Trypan blue staining revealed the nature and extent of fungal hyphae colonization with C graminicola initially invading via thick primary hyphae and later switching to a necrotrophic stage.

C graminicola invades living maize cells using thick primary hyphae separated from the host cytoplasm by a membrane. In contrast, S matus produces a phytotoxin that kills the epidermal cells before invading them. In C graminicola, the production of dark brown precipitate in the maize leaf sheaths indicated an oxidative burst related to a host resistance response.