RNA extraction, RNA-seq and quantitative real-time PCR analysis
Plant materials were collected after 4 d co-cultivation. Frozen samples of the control and SQR9 VCs-treated seedlings were ground in liquid nitrogen and total RNA was extracted with three biological replicates resulting in a total of 6 samples. Experimental protocols for RNA sequence was performed according to the manufacture’s (Novogene Experimental Department) technical instruction.
Differential expression analysis of control and SQR9 VCs-treated samples was performed using the DESeq R package (1.18.0). DESeq provide statistical routines for determining differential expression in digital gene expression data using a model based on the negative binomial distribution. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. Genes with an adjusted P-value < 0.05 found by DESeq were assigned as differentially expressed. The auxin-related differentially expressed genes with a |log2(FoldChange)| > 1 were selected for further analysis.
Three-day-old Arabidopsis seedlings of Col-0 grown on MS slats agar medium were exposed to SQR9 VCs or not. After 4 d of co-cultivation, total RNA was isolated from control or SQR9 VCs-treated seedlings using Plant RNA Kit R6827 (OMEGA bio-tek) following the manufacturer’s protocol. The first-strand cDNA synthesis was generated from 1 μg of total RNA using the HiScript 1st Strand cDNA Synthesis Kit (Vazyme). Quantitative RT-PCR was performed on a StepOnePlus™ Real-Time PCR System (Applied Biosystems) using an SYBR® Green Master Mix Kit (Vazyme).AtEF1A (AT5G60390) was used as the reference gene and the primers are listed in Supplemental Table S2. The results were obtained from three biological replicates and 2–ddCt was taken for every sample as the relative expression levels. Values detected by quantitative RT-PCR are relative to the lowest value for each gene.