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).