Salicylic acid and methyl jasmonate treatments

48 h after agro-injection (Liu et al. 2011; Yang et al. 2000), 2 mM of salicylic acid and 50 μM of methyl jasmonate (Shokouhifar et al. 2011a) both from Sigma Aldrich (Darmstadt, Germany) were separately sprayed on agro-injected leaves for each tobacco species. Two tobacco plants from N. tabacum cv. Xanthi and Nicotiana benthamiana species were used without any agro-injection as salicylic acid control. Two plants were also selected for methyl jasmonate control. The control plants were separately sprayed with salicylic acid and methyl jasmonate. Leaf disks were sampled for GUS assay 24 h after treatment with salicylic acid and methyl jasmonate (Yang et al. 2000). Leaf discs imaging was carried out after histochemical staining and chlorophyll removal procedures using a digital microscope (Dino-Lite Am-313 model T Plus made in Taiwan).