Gene expression analysis
Roots and shoots (from cotyledons upwards) were collected for mRNA
sequencing 14 days after transplantation (one day after second PA
treatment). For RT-qPCR, roots and shoots were harvested 6, 24 and 72 h
after the second PA treatment. Samples were ground in a liquid-nitrogen
chilled mortar.
For mRNA sequencing, RNA was extracted from ±100 mg ground tissue
(Quick-RNA Plant Miniprep Kit, Zymo Research). Three biological
replicates, each consisting of four pooled plants, were used per
treatment. Library preparation, sequencing and data analysis were as in
(Ghaemi et al. 2020). Briefly, libraries were prepared using the
QuantSeq 3’ mRNA-Seq library prep kit (Lexogen), library quality was
verified using an Agilent Bioanalyzer 2100 and libraries were sequenced
on an Illumina NextSeq 500 platform. Data quality was assessed using
FastQC (Andrews 2010), reads were trimmed with Trimmomatic (Bolger,
Lohse & Usadel 2014; 5 bp sliding window, bases with Phred scores
< 20 were trimmed and reads < 40 bp were removed).
Trimmed reads were mapped to the tomato reference genome (ITAG 4.0,
Hosmani et al. 2019) using STAR (Dobin & Gingeras 2015) and
reads were counted using the summarizeOverlaps function of the RGenomicAlignments package (Lawrence et al. 2013).
Differentially expressed genes were identified using the R DeSeq2package (Love, Huber & Anders 2014). Genes were considered
differentially expressed if FDR-adjusted P < 0.05 and
|log2 FC| > 0.7. PLAZA
4.5 (Van Bel et al. 2018) was used for gene ontology analysis.
For RT-qPCR, RNA was extracted from ±100 mg ground tissue (RNEasy Plant
kit, Qiagen,), DNAse-treated (DNAse I, Thermo Fisher Scientific) and
converted to cDNA (Tetro cDNA synthesis kit, Bioline). RT-qPCR
conditions were as in De Kesel et al. (2020). Primer sequences
are listed in Supplementary Table S1 . Three reference genes
were used, using primer sequences obtained from Cheng et al. (2017).
Reference gene stability in shoot and root was verified using our
mRNA-seq dataset. Amplification efficiency and relative expression were
calculated using LinRegPCR v. 2020.0 (Ruijter et al. 2009).
Statistical significance was determined using FDR-adjusted t-tests. Four
biological replicates, consisting of 2-3 pooled plants, were used per
treatment.