Gene Expression Analysis of Salicylic and Jasmonic Acid Pathways

The saliva collection and infiltration treatments were as described in Section 2.3. The leaves were collected 6 h and 24 h after infiltration and stored at −80 °C until use. Total leaf RNA was extracted using TRIzol reagent (Tiangen, Beijing, China), and 1 μg RNA was used to synthesize the first cDNA strand with FastQuant cDNA (Tiangen, Beijing, China) according to the manufacturer’s instructions. Real-time quantitative-polymerase chain reaction (RT-qPCR) was performed in 20 μL reaction volumes containing 10 μL 2× SYBR Premix (Tiangen, Beijing, China), 8 μL water, 1 μL gene-specific primers, and 1 μL cDNA template. The reaction was performed with a CFX Connect^TM Real-Time PCR System (Bio-Rad, Hercules, CA, USA). The temperature protocol was: 95 °C for 30 s, followed by 40 cycles at 95 °C for 10 s, 60 °C for 20 s, and 72 °C for 30 s. The expression of five genes involved in the SA pathway and five genes involved in the JA pathway were detected. CitGAPC1 was used as an internal control. Sequences and description of primers are listed in Table 1 [34 (link),35 (link),36 ,37 (link)]. There were four replicates per treatment (each leaf was considered a replicate), and each replicate contains three technique replicates.