*4.4. RNA Isolation and Quantitative Real-Time PCR*

Total RNA of detached leaves from DIS was isolated using NucleoSpin RNA (Macherey Nagel, Deuren, Germany), according to the manufacturer's instructions. Following reverse-transcription into the first-strand cDNA with SmartGene Mixed cDNA synthesis kit (SJ Bioscience, Daejeon, Korea), real-time PCR was performed using a CFX Connect Real-Time System (Bio-Rad, Hercules, CA, USA). Each reaction contained 10 μL of 2× SYBR Green Master Mix reagent, 2 μL of diluted cDNA samples, 1 μL of 10 pmol gene-specific primers in a final volume of 20 μL. The real-time PCR protocol was: 15 min at 95 ◦C to denature and activate an enzyme, followed by 40 cycles at 95 ◦C for 20 s (denaturation), 60 ◦C for 40 s (annealing), 72 ◦C for 30 s (extension). qRT-PCR was conducted according to the 2-ΔΔCt method [48]. Rice *UBIQUITIN5* (*OsUBQ5*) was used for normalization, and relative expression levels were compared by Student's *t*-test. Primers used in this study are listed in Table S1.
