2.8 Quantitative PCR
Total RNA was isolated as described above. 1 µg of total RNA was reverse-transcribed using the HiScript Reverse Transcriptase (Vazyme, Nanjing, China). Quantitative PCR was performed using Hieff UNICON® Universal Blue qPCR SYBR Green Master Mix (YEASEN, Shanghai, China) (Laveroni & Parks, 2023) according to the manufacturer’s protocol and run in a LightCycler 480 II Real-Time System R (Roche Diagnostics, Basel, Switzerland). We calculated transcript levels of treatment group relative to the control group using the 2-ΔΔCtmethod (Livak & Schmittgen, 2001) with normalization based on the reference gene OsEF1-alpha (Bevitori et al., 2014). The qPCR was repeated three times, with two samples per replicate, and the qPCR primers are listed in Table S23.
2.9 Phytohormones, cellulose, and hemicellulosequantification
DRS-sequenced samples were collected for JA, JA-IlE, and SA content measurement. The fresh weight tissue samples were ground to powder in liquid nitrogen, extracted using ethyl acetate spiked with labeled internal standards (2D6-JA,2D6-JA-IlE, and 2D4-SA), and analyzed by liquid chromatography-tandem mass spectrometry (HPLC-MS) for phytohormones quantification as previously described (Ji et al., 2021).
For cellulose and hemicellulose quantification, twenty BPH female adults were placed on each rice leaf sheath and continuous feeding for 24 hours. Then, the BPHs were removed and rapid stem sample collection within the feeding region was conducted. The methods of extraction and measurement of cellulose and hemicellulose fractions were previously described (Guo et al., 2018).