4.5. RNA extraction and gene expression analysis by RT-qPCR

RNA extraction was carried out from the rice leaves using TRIzol solution (Invitrogen, Carlsbad, U.S.A), followed by the synthesis of first-strand cDNA from 2 µg of the isolated RNA using M-MLV reverse transcriptase (Promega, Madison, USA). Real-time quantitative PCR (RT-qPCR) was conducted in triplicate using a Rotor-gene Q instrument (Qiagen, Hilden, Germany) and Prime Q-Mater Mix (Genetbio, South Korea), following the manufacturer's instructions. The specific primers utilized are listed in Supplementary Table S5. To determine the relative fold differences in template abundance for each sample, the threshold cycle (Ct) value for each gene of interest was normalized to the Ct value of OsUbi5 and calculated relative to a calibrator using the 2-ΔCT algorithm.