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].