Accuzol

Total RNA was isolated using AccuZol (Bioneer), and first strand of cDNA was synthesized by reverse transcribed to cDNA using a ReverTra Ace qPCR kit (Toyobo, Osaka, Japan). A quantitative real-time PCR was performed to validate the expression level using SYBR^® Premix Ex TaqTM (TaKaRa Bio, Shiga, Japan) with specific primers (Table 1) on Applied Biosystems 7300 machine (Applied Biosystems, Carlsbad, CA, USA). The relative values for specific mRNA were calculated after normalization to the Ct value from β-actin in the same sample using the DDCt method.

Three independent biological replicates of 15 Norang leaf discs (100 mm²) treated with water, 100 μg ml⁻¹ chitin or 100 nM flg22 for 30 and 180 min were generated. Total RNA was extracted using Accuzol (Bioneer, Korea) and treated with DNAse I. RNA samples were quantified and the purity assessed by Agilent chromatography. Libraries of mRNA were constructed from 1 μg total RNA using TruSeq Stranded mRNA LT Sample Prep kit and sequenced using Illumina NovaSeq 6000 sequencing system by Macrogen (Korea). Low quality reads were trimmed using Trimmomatic. The trimmed reads were mapped to B. rapa reference genome (Bra chromosomev1.5; brassicadb.org/brad/datasets/pub/BrassicaceaeGenome/Brassica_rapa/V1.0/Bra_Chromosome_V1.5/) using HISAT2. Read count and FPKM were obtained from transcript assembly using StringTie. Differentially expressed genes were called using DESeq2 with log2 fold-change threshold of 2 and nbinomWald Test raw p-value < 0.05. Hierarchical clustering of glucosinolate biosynthesis genes was performed using Pearson distance and average linkage in Heatmapper^{49 (link)}. The transcriptomic data can be found in the Gene Expression Omnibus repository under the accession number GSE150746.

Norang or N. benthamiana leaf discs (100 mm²) were floated overnight on deionized water then treated with different MAMP solutions (50 μg ml⁻¹ LPS, 50 μg ml⁻¹ PGN, 100 nM elf18, 100 μg ml⁻¹ chitin or 100 nM flg22) or water for 30, 60, and 180 min. Total RNA was extracted using Accuzol (Bioneer, Korea) according to the manufacturer’s instructions. RNAs were treated with DNAse I to remove residual genomic DNA and 1 μg total RNA was used for cDNA synthesis with the Maxima first-strand cDNA synthesis kit (Thermofisher). For quantitative RT-PCR, cDNA template was combined with GoTaq qPCR master mix (Promega) and PCRs were performed in triplicate with a LightCycler480 system (Roche) with the primers listed in Table S4.

Total RNA was isolated using AccuZol (Bioneer), and the first strand of cDNA was synthesized by reverse transcription using a ReverTra Ace qPCR kit (Toyobo, Osaka, Japan). qRT-PCR was performed to validate the expression levels using SYBR Premix Ex TaqTM (TaKaRa Bio, Shiga, Japan) with specific primers (Table 1) in an Applied Biosystems 7300 machine (Applied Biosystems, Carlsbad, CA, USA). The relative values for specific mRNA were calculated after normalization to the Ct value from β-actin in the same sample using the ddCt method.