Quantitative real-time PCR (qPCR) and enzyme-linked immunosorbent assay (ELISA)
Total RNA was extracted from inoculated leaves and young leaves using RNA extraction kit (Promega Biotechnology Co., Ltd., Shanghai, China). Next, the reverse transcription kit (Shanghai Titan Technology Co., Ltd., Shanghai, China) was used to synthesize cDNA. Quantitative PCR (qPCR) was performed according to the previous method using a CFX Touch Real‐time PCR machine (Bio‐Rad, USA) and Quantinova™ SYBR Green PCR Kit (Chongqing Shuguang Biotechnology, China) [66].Actin was used as an internal reference. Quantification of the relative changes in gene transcript levels was performed using the 2-ΔΔCT method. Each sample has three biological replicates. All primers of qPCR were listed in Table 1 and S1 . The crude extracts prepared from leaf samples at different inoculation times were used to perform ELISA assay. TMV-GFP antibodies and horseradish substrate coloration were used to detect the protein accumulation of TMV-GFP [29-30].