Live-cell Imaging of Fungal Cells to Investigate Modes of Entry and Subcellular Localization of Antifungal Plant Defensins

The overall goal of this live-cell imaging method is to study critical aspects of the mechanisms of action of antifungal plant defensins in fungal cells. With the advent of advanced confocal microscopy techniques, live-cell imaging has become a powerful tool to study the modes of action of antifungal plant defensins. Making use of this technology, here we present a method to help answer key question in understanding the modes of action of antifungal plant defensins.

Our focuses are how these peptides are internalized into the fungal cells and how these peptides are localized into the cell organelles or diffused in the cytoplasm. To make a conidial suspension of PH-1 strain of Fusarium graminearum, first set up cultures of the strain on plates containing complete medium. Culture them at 28 degrees Celsius over five days.

To produce conidia, inoculate four 10-millimeter diameter plugs of the five-day-old culture into 50 milliliters of carboxymethyl cellulose medium. Culture these plugs for four to seven days at 28 degrees Celsius on a rotary shaker set to 180 rpm. When the cultures have a red color, the conidia have formed.

To collect the conidia in a suspension, vortex the liquid culture, and filter one milliliter of it through two layers of filtration material, into a two-milliliter microcentrifuge tube. Centrifuge the suspension for two minutes, and discard the supernatant. Then, wash the pellet with one milliliter of sterile water, and repeat the centrifugation.

Next, resuspend the pellet in one milliliter of 2x SFM. Then, count the conidia using a hemocytometer, and adjust the density of the suspension to 100, 000 conidia per milliliter. To make a Neurospora crassa conidial suspension, transfer conidia from stock cultures to a slant tube containing Vogel's agar medium, and incubate the tubes at room temperature under constant light for five days.

After five days, transfer a small amount of growing culture, using an inoculation loop, to a microcentrifuge tube containing two milliliters of Vogel's liquid medium. Be sure to mix the conidia off the loop. Then, filter the suspension, centrifuge it, and resuspend the pellet of conidia in Vogel's medium without a wash step.

Finally, adjust the final suspension to 100, 000 conidia per milliliter. To make a germlings preparation for confocal microscopy, pipette 50 microliters of conidia suspension onto a 35-millimeter culture dish, and let the conidia germinate for three to six hours at room temperature. For confocal microscopy, pipette 50 microliters of conidial suspension into a 10-millimeter microwell of glass-bottom dish.

Then, at the predetermined minimal inhibitory concentration, add 50 microliters of fluorescently labeled defensins to the suspension, and incubate the conidia with the labeled defensins for 2.5 hours at room temperature. After the incubation, add two microliters of the membrane selective dye FM4-64, for a final concentration of five micromolar. Then, incubate the culture for 30 minutes at room temperature prior to examining the conidia.

To set up the confocal microscope, select the white light laser. Use the 488-nanometer and 550-nanometer lasers to excite the rhodamine-labeled defensins and the FM4-64 dye, respectively. Set these lasers to an intensity of 1%Then, set the detection wavelengths to 580 to 700 nanometers for the rhodamine-labeled defensin and from 690 to 800 nanometers for the FM4-64 dye.

Now, collect the images. For time-lapse imaging, add 50 microliters of conidial suspension into a glass-bottom microwell dish. Next, mount the microwell dish on the microscope, and find the cells under low power.

Then, switch to a 100x, 1.44 oil objective. To observe the DyLight550-labeled defensins, set the laser line to 550 nanometers for excitation and from 560 to 600 nanometers for detection. Next, set the scan mode to xyzt.

Then, set the Z-position, the zoom, the frequency of image capture, and so forth, as needed. Now, to the conidial suspension, add 50 microliters of fluorophore-labeled defensins at the minimal inhibitory concentration of three micromolar, and add two microliters of membrane selective dye FM4-64 for a final concentration of five micromolar. Then, refocus the optics if needed.

Before capturing images, place small pieces of wet filter paper in the microwell dish to prevent evaporation. Then, process with capturing an image every 3.5 minutes over 2.5 hours. Live-cell imaging was carried out to track and compare the internalization and subcellular localization of two defensins from Medicago truncatula.

Chemically synthesized rhodamine-labeled defensin four was trafficked differently in Neurospora crassa and Fusarium graminearum. FM4-64 labeled the plasma membranes of both fungi. However, in Fusarium, defensin four is diffused in the cytoplasm.

Whereas, in Neurospora, it is transported to vesicular bodies. For comparison, defensin five was visualized using a DyLight550 label in Neurospora crassa, along with plasma membrane labeling by FM4-64. Time-lapse imaging shows that this defensin enters the cells within 30 to 40 minutes and then diffuses through the cytoplasm.

This differs from the trafficking of defensin four, which enters the cells and remains trapped within the vesicular bodies even after three hours. In summary, live-cell imaging is a powerful tool to increase our understanding of the modes of action of antifungal plant differences. With other vital fluorescent dyes and cellular markers, it has the flexibility to be modified for other antimicrobial peptides.

Ultimately, this technique can help to develop new strategies for use of antimicrobial peptides as an antifungal agent in agriculture and medicine.