RNA Extraction and Quantitative RT-PCR (qRT-PCR) Analysis

Leaves of 4-week-old Arabidopsis (∼0.1 g) plants were harvested at the indicated time points after Pseudomonas inoculation. Total RNA was extracted from the samples using TRIzol reagent (Thermo Fisher Scientific), and contaminating genomic DNA was removed using TURBO DNase (Ambion). First-strand cDNA was synthesized from 5 μg of total RNA using SuperScript-II Reverse Transcriptase (Thermo Fisher Scientific) according to the manufacturer’s instructions. Quantitative PCR (qPCR) was performed using SYBR Premix Ex Taq (TaKaRa Bio) and a CFX384 Real-time PCR Detection System (Bio-Rad). The cycling conditions were 95°C for 10 min, and 50 cycles of 95°C for 5 s, 60°C for 10 s, and 72°C for 35 s. ACTIN2 (At3g18780) was used as the reference gene to normalize transcript levels. Relative expression levels were analyzed using the comparative cycle threshold (ΔΔCt) method (Livak and Schmittgen, 2001) and are shown as mean ± SD (standard deviation). All experiments were performed with at least three biological replicates with two or three technical repeats unless otherwise noted. Asterisks indicate statistically significant differences from WT plants (^∗p < 0.05, ^∗∗p < 0.01, two-tailed Student’s t-test). The oligonucleotide sequences of the primers are shown in Supplementary Table 2.