FIGURE 3.

ZmGABA-T suppresses allergic necrosis induced by EG1 according to promoting the degradation of EG1, rather than inhibit the enzyme activity of EG1. (A) Transient expression of EG1 and ZmGABA-T in Nicotiana benthamiana. Transient expression was operated in Nicotiana benthamiana leaves injected with Agrobacterium carrying the indicated genes, pGR106, eGFP, EG1, and ZmGABA-T. eGFP and EG1 were coexpressed on the left of the leaf, and ZmGABA-T and EG1 were coexpressed on the right. Experiments were repeated three times at least with similar results. (B) ROS detection of EG1 and ZmGABA-T. The transient expression of Nicotiana benthamiana leaves inoculated with eGFP and EG1, ZmGABA-T and EG1 were dyed with 5 mg/mL DAB (3,3-diaminobenzidine). Transient expression was assessed in Nicotiana benthamiana leaves from 4-week-old plants 5 days after agroinfiltration, and pGR106 empty vector was used as a control. Experiments were repeated three times at least with similar results. (C) Enzyme activity determination of EG1 and ZmGABA-T. Co-incubation EG1 (1 μM) expressed by Pichia pastoris GS115 with pGR106 and ZmGABA-T expressed by Nicotiana benthamiana (2 mg/mL) separately at 50°C for 30 min, then dinitrosalicylic acid (DNS) was used to determine glucose content, and measured absorbance at OD₅₄₀, thereby calculated enzyme activity of EG1. The results of the experiment were repeated three times at least. Lower-case letters indicate statistically significant differences (P < 0.05). (D) Co-expression of EG1-eGFP and ZmGABA-T-HA in Nicotiana benthamiana. The concentration of inoculation about ZmGABA-T was increased sequentially from OD₆₀₀ = 0 to 0.9, while the concentration of EG1 was OD₆₀₀ = 0.5. The numbers in the first row indicated the inoculation concentration of ZmGABA-T-HA. The numbers in the second row indicated the gray value of EG1. The results of the experiment were repeated three times at least.