Brassinazole

Brassinolide (BL, the most active BR) and brassinazole (BRZ, a specific inhibitor of BR biosynthesis) were purchased from Wako Pure Chemical Industries, ltd (Chuo-Ku, Osaka, Japan) and Santa Cruz Biotechnology, inc (Dallas, Texas, USA), respectively. Bikinin was purchased from Sigma (St. Louis). The hormone and inhibitor solutions were prepared in water containing 0.02% (vol/vol) Tween 20. The chemicals and the concentrations used are as follows: BL (0.1 μM), BRZ (1 μM) and Bikinin (50 μM). Distilled water containing 0.02% (vol/vol) Tween 20 was used as a control treatment.
In infection experiments, the chemicals were sprayed 12 h before virus inoculation. Purified TMV-GFP RNA was maintained in an aqueous suspension of 0.02 M sodium phosphate buffer (PBS) at 4 °C. Three leaves of each N. benthamiana plant were inoculated with 0.1 μg of TMV-GFP RNA. PBS buffer without virus RNA was rubbed onto the leaves as the control experiment.

Brassinolide (BL, the most active BR) and brassinazole (BRZ, a specific inhibitor of BR biosynthesis) were purchased from Wako Pure Chemical Industries (Chuo-Ku, Osaka, Japan) and Santa Cruz Biotechnology (Dallas, Texas, USA), respectively. 1-Aminocyclopropane-1-carboxylic acid (ACC; precursor of the ethylene biosynthesis) and ethylene biosynthesis blocker aminoethoxyvinylglycine (AVG) were purchased from Sigma-Aldrich (Shanghai, China). Dimethylthiourea (DMTU, an H₂O₂ scavenger), 1-methylcyclopropene (1-MCP) and diphenylene iodonium (DPI, an NADPH oxidase inhibitor) were purchased from Sigma (St Louis, USA). The hormone and inhibitor solutions were prepared in distilled water containing 0.02% (v/v) Tween 20. The chemicals and the concentrations used are as follows: BL, 0.01, 0.1, 1, and 5 μM; BRZ, 1 μM; DMTU, 5 mM; DPI, 100 μM; AVG, 5 μM. ACC, 10 μM. Distilled water containing 0.02% (v/v) Tween 20 was used as a control treatment. For DMUT/DPI + BL treatment, plants were first sprayed with 5 mM DMTU/100 μM DPI, and 8 h later they were sprayed with 0.1 μM BL for another 12 h. For BL + AVG or BRZ + ACC treatment, plants were first sprayed with 5 μM AVG or 10 μM ACC, and 8 h later they were sprayed with 0.1 μM BL or 5 μM BRZ for another 12 h. The plants were then exposed to 200 mM NaCl.

Brassinolide (BL, the most active BR) and brassinazole (BRZ, a specific inhibitor of BR biosynthesis) were purchased from Wako Pure Chemical Industries (Chuo-Ku, Osaka, Japan) and Santa Cruz Biotechnology (Dallas, Texas, USA), respectively. Salicylhydroxamic acid (SHAM, an inhibitor of the AOX pathway) and dimethylthiourea (DMTU, an H₂O₂ scavenger) were purchased from Sigma (St Louis, USA). The hormone and inhibitor solutions were prepared in distilled water containing 0.02% (v/v) Tween 20. The chemicals and the concentrations used are as follows: BL, 0.01, 0.1, 1, and 5 μM; BRZ, 1 μM; SHAM, 1mM; DMTU, 5mM. Distilled water containing 0.02% (v/v) Tween 20 was used as a control treatment. For SHAM+BL treatment, plants were first sprayed with 1mM SHAM, and 8h later were sprayed with 0.1 μM BL for another 24h. For DMTU+BL treatment, plants were first sprayed with 5mM DMTU, and 8h later were sprayed with 0.1 μM BL for another 24h. The plants were then exposed to environmental stress.