Evaluating FocCP1 Elicitor Function in Tobacco

To analyze whether FocCP1 exhibited elicitor function, 4-week-old tobacco leaves were infiltrated with FocCP1 (100 μM) with BSA (100 μM) as a control. The samples were cryo-preserved in liquid nitrogen post-treatment of 8, 16, and 24 h, three biological replicates were performed [15 (link)]. All total RNA was extracted with the RNA Prep-Pure Plant Kit (TianGen, Beijing, China) according to the manufacturer’s protocol. First-strand cDNAs were synthesized with the same method as in Section 4.4. Six defense-related genes, PR1, PR5, PAL, EDS1, HSR203J, and LOX, were amplified using an ABI 7500 Real-Time PCR system (Applied Biosystem, Foster CA, USA) with Trans Start^® Green qPCR Super Mix UDG (TransGen Biotech, Beijing, China). Tobacco actin gene was the endogenous control and the qRT-PCR primers are listed in Table 3 [67 (link)]. The amplification program was as follows: 50 °C for 2 min, 94 °C for 10 min, followed by 40 cycles of 94 °C for 5 s, 60 °C for 15 s, and 72 °C for 15 s. The relative mRNA expression values were calculated according to 2^−ΔΔCt methods [68 (link)]. For each gene, qRT-PCR assays were repeated three times with three biological replicates each time.