Trypan Blue Staining, Oxygen Burst, and Callose Deposition Detection

For detection of dead cells, tobacco leaves were stained with trypan blue (Fernández-Bautista et al., 2016). Briefly, agroinfiltrated leaves at 5–7 days postinfiltration (dpi) were collected and immersed in ethanol: glacial acetic acid solution (3:1, v/v) for 24 h to fix the cell tissues and destain chlorophyll. The treated leaves were then soaked in a solution containing trypan blue dye [10 ml lactic acid, 10 ml glycerin, 10 ml deionized water, 10 g phenol, and 20 mg trypan blue (Sigma-Aldrich, St. Louis, United States)] for 24 h. Tissue images were taken and decolorization was performed. Samples were decolorized in 1.25 g/ml chloral hydrate solution for 3 days. Triplicated bioassays were performed to confirm the reproducibility of the effector function. Oxygen burst status in tobacco leaves was monitored by detecting accumulation of H₂O₂ using 3,3′-diaminobenzidine (DAB) according to the method of Thordal-Christensen et al. (1997). Briefly, agroinfiltrated tobacco leaves were detached at 72 h postinfiltration (hpi), soaked in 1 mg/ml DAB solution, and incubated at 25°C for 8 h. Leaf tissues were then boiled in 95% ethanol until all chlorophyll pigments were entirely bleached. For further removal of the background color, the bleached samples were soaked in 1.25 g/ml trichloroacetic aldehyde, and the leaves were photographed. Callose deposition was monitored at 48 h after agroinfiltration of N. benthamiana. All experiments were repeated at least three times.