RNA-Seq analysis of genes affected by Cyclophilin A/DIAGEOTROPICA (DGT) in tomato root development

Plant material and growth conditions

Three biological replicates for each tissue and genotype were performed. Seven-day old tomato (Solanum lycopersicum) wildtype (WT) and dgt^1-1 plants in the ‘Ailsa Craig’ background⁷ were used. Seeds were sterilized in 20% commercial bleach for 30 min and rinsed four times for 10 min with sterile water. Sterilized seeds were vernalized at 4°C for two days to ensure even germination and then planted on media containing 0.2× Murashige and Skoog basal medium with vitamins (PhytoTechnology), 1% sucrose, 10 mM MES buffer pH 5.7, and 0.8% agar. Seeds were germinated in Magenta boxes (16 seeds per box) in a growth chamber at 21°C under long day (16h light, 8h dark) conditions and light intensity as in Ivanchenko et al. (2013). Root samples were dissected under DIC optics at 4x objective as in Ivanchenko et al. (2006). On average, 50–100 root portions were collected per biological replicate. The meristem was dissected between the root tip and the root portion where tissue becomes more transparent. The elongation zone was collected from the proximal meristem border and the first hair bulge, and approximately 1 cm portions were collected above the first hair bulge for the differentiation zone.

RNA isolation and RNA-Seq

Tissue samples were collected in Plant RNA Reagent (Life Technologies) on ice, and total RNA was prepared using the RNeasy Mini kit (Qiagen) according to manufacturer’s recommendations. The RNA pellets were dried in 1.7 mL centrifuge tubes and solubilized in 178 µL 1X RNA Secure Reagent (Ambion) preheated at 65°C, and incubated at 65°C for 10 min, mixing by pipetting a few times. Then 20 µL 10X DNase I buffer and 2 µL RNase-free DNase I (Ambion) were added to each tube, and tubes incubated for 10 min at 37°C. 700 µL RLT buffer was added (to which 7 µL 2-Mercaptoethanol was freshly added) to each sample, which were then mixed by vortexing. 500 µL ethanol was then added and samples were mixed again by vortexing. Each sample (2 X 700 µl) was applied to an RNeasy Mini spin column from the RNeasy kit and RNA cleanup performed following the manufacturer’s instructions. Each sample was eluted with 30 µL nuclease-free ultrapure H₂O, and the RNA concentrations were measure using a NanoDrop 1000 spectrophotometer (Thermo Fisher).

Illumina sequencing of RNA

RNA-Seq library preparation and sequencing were performed at the Oregon State University Center for Genome Research and Biocomputing. Libraries were prepared using the TruSeq RNA Library Prep Kit v2 (Illumina) and sequenced as single-end 51 bp reads on the Illumina HiSeq 2000 using a total of two lanes.

Gene alignment and expression level analysis

Sequencing produced 5-8 FASTQ files per replicate, which were merged into a single FASTQ file per replicate. Reference transcripts were extracted and preprocessed from the NCBI Heinz 1706 genome sequence¹⁵ using RSEM’s¹⁶ (RNA-Seq by Expectation Maximization, version 1.3.1) rsem-prepare-reference function. Using RSEM, raw sequence reads were then aligned to the reference transcript sequences and estimated transcripts per million (TPM) values were calculated using default parameters. Resulting gene-level estimates from biological replicates were merged into a single input matrix and EBSeq¹⁷ (version 1.26.0) was then used to test for differential expression between dgt and WT for each root-tip zone. Raw RNA-Seq reads, abundance estimates, and differential expression analysis are publicly available in the Sequence Read Archive (SRA) and Gene Expression Omnibus (GEO), see Data availability.

Data visualization

As a preliminary analysis to guide future investigations, a basic comparison of presence/absence calls and differential gene expression is presented. Expression analysis was performed using TPM and differential expression analysis was performed using the posterior probability that the gene is differentially expressed (PPDE) and posterior fold change (postFC). Genes with an average of TPM > 2 across biological replicates were compared between zones and genotypes (Figure 1; data summarized in Data File 1¹⁸). Following EBSeq analysis, genes were filtered for PPDE = 1 to identify those which were differentially expressed. Of the differentially expressed genes, those with postFC (dgt over WT) > 2 were considered upregulated in dgt and those with postFC < 0.5 were considered downregulated in dgt. Upregulated and downregulated genes were compared between zones and genotypes (Figure 2; data summarized in Data File 2¹⁹). Principal component analysis (PCA) of the data was performed using TPM values of each gene and was calculated using the scikit-learn²⁰ PCA function with default parameters (Figure 3).

Figure 1. Presence/absence calls.

(A) Number of genes expressed in each zone of dgt roots, transcripts per million (TPM) > 2.0. (B) Number of genes expressed in each zone of WT roots, TPM > 2.0. (C) Number of genes expressed exclusively in the differentiation zone with TPM > 2.0, compared between WT and dgt. (D) Number of genes expressed exclusively in the elongation zone with TPM > 2.0, compared between WT and dgt. (E) Number of genes expressed exclusively in the meristem with TPM > 2.0, compared between WT and dgt.

Figure 2. Differentially expressed genes.

(A) Differentially expressed genes downregulated in each zone with postFC < 0.5, posterior probability that the gene is differentially expressed (PPDE) = 1 in dgt vs WT. (B) Differentially expressed genes upregulated in each zone with postFC > 2, PPDE = 1 in dgt vs. WT.

Figure 3. Principle component analysis (PCA).

(A) PCA of sequenced samples in differentiation zone (B) PCA of sequenced samples in elongation zone (C) PCA of sequenced samples in meristem.

Source data

Heinz 1706 genome available from Assembly, Accession number GCF_000188115.4: https://www.ncbi.nlm.nih.gov/assembly/GCF_000188115.4/

Underlying data

Raw RNA-Seq reads and expression estimates provided by RSEM on Gene Expression Omnibus, Accession number GSE156398: https://identifiers.org/geo:GSE156398

Figshare: Data File 1: Genes referenced in figure 1. https://doi.org/10.6084/m9.figshare.12891773.v1¹⁸.

Figshare: Data File 2: Genes referenced in figure 2. https://doi.org/10.6084/m9.figshare.12891941.v1¹⁹.