<p>All samples were imaged using fluorescent scanning confocal microscopy (Nikon confocal A1R, excitation 488-nm@16mW, emission filter 525/50) to evaluate fusion events. Cells were segmented using the transmitted light channel and instances of bright fluorescent spots within cells were evaluated. Image analysis was performed in ImageJ.</p>
<li>Grow yeast cells in YPD medium overnight.</li>
<li>Harvest the cells and wash twice with sterile water, then twice with 1 M sorbitol solution (20 mL per gram of cells).</li>
<li>Resuspend the cells in sterile-filtered 1 M sorbitol solution (5 mL per gram of cells) containing Zymolyase 100T (5-10 mg/g of cells).</li>
<li>Incubate the suspension in a water bath at 37°C for 1 hour to generate spheroplasts.</li>
<li>Cool the spheroplast suspension on ice for 20-30 minutes, then centrifuge at 1,500g, 4°C for 10 minutes.</li>
<li>Wash the spheroplasts twice with 1 M sorbitol solution (5 mL per gram of cells) and resuspend in 1 M sorbitol solution (2 mL per gram of cells).</li>
<li>Mix 500 µL of spheroplast suspension with 500 µL of TSC buffer, incubate at 30°C for 10 minutes, then centrifuge at 1,500g for 10 minutes.</li>
<li>Resuspend the spheroplasts in 100 µL of TSC buffer.</li>
<li>Mix 150 µL of bacterial cell suspension quickly with 50 µL of 4 M sorbitol, then immediately add the mixture to 100 µL of spheroplast suspension in TSC buffer.</li>
<li>Invert the tubes to mix and incubate at 30°C for 10 minutes.</li>
<li>Add the mixture to 2.5 mL of PEG buffer and incubate at 30°C for 45 minutes.</li>
<li>Centrifuge at 1,500g, 25°C for 10 minutes. Discard the supernatant PEG solution.</li>
