Fig. 1
Global profiling of alternative splicing events in response to flg22 treatment. A Scheme of in-depth transcriptome profiling for alternative splicing in response to flg22. Two-week-old plate-grown wild-type (WT) Col-0 and cpl3-3 mutant seedlings treated with H2O (mock) or 100 nM flg22 for 60 min were subjected to RNA isolation and sequencing. To capture the transcripts with low abundance, RNA-seq was performed with Illumina HiSeq 2500 to obtain 120 million reads per sample with the paired-end 150-bp read length, which is about 530 × coverage of Arabidopsis transcriptome. The sequenced reads were aligned to the AtRTD3 reference transcriptome for quantification. The 3D RNA-seq analysis was performed to identify differentially expressed genes (DEGs), differentially alternatively spliced genes (DASs), and transcripts with differential transcript usage (DTUs). The bottom left panel shows an example of DEGs with two differentially expressed transcripts, where changes in abundance between conditions (dH2O and flg22 treatments) are measured by log2 fold change. Total gene expression is represented in the blue line, which is the sum of the expression of all individual transcripts (green and yellow lines). The percentage values denote the expression ratios of individual transcripts relative to the total gene expression. The bottom right panel illustrates examples of DAS and DTU. For a DAS gene, it must have more than one transcript, and changes in expression between individual transcripts (green, yellow, and purple lines) and the total gene expression (blue line) are compared between conditions. The change in percent spliced (ΔPS) is the percentage change in the abundance of a transcript relative to the total gene expression. For a gene to be classified as DAS, at least one transcript has a |ΔPS|≥ 0.1. In DTU analysis, individual transcripts show different expression patterns compared to other transcripts of the same gene. DTU is identified by comparing the change in expression of each transcript to the average expression change of the other transcripts within the same gene. In this example, the transcripts represented with green and yellow lines, but not with the purple line, are DTUs. B Flg22 treatment triggers transcriptional changes in gene expression and alternative splicing in WT plants. The Y-axis indicates the numbers of flg22-triggered DEGs and DAS genes, and DTU transcripts. The flg22-triggered up-/downregulated DEGs were identified based on an absolute value of fold change (|FC|) ≥ 2 and false discovery rate (FDR) < 0.01 between mock and flg22 treatment. The flg22-triggered DASs and flg22-triggered DTUs were selected based on an absolute delta percent spliced (|ΔPS|) ≥ 0.1 and FDR < 0.01 between mock and flg22 treatment. C Volcano plot of flg22-triggered DTUs in WT. Up- and downregulated DTUs in response to flg22 in WT were depicted by a volcano plot. Purple and pale purple dots represent up- and downregulated flg22-DTUs, respectively. The DTUs with non-statistically significant differences were indicated as gray. The Y-axis denotes − log10(FDR), while the X-axis shows ΔPS values. The cut-off lines for FDR = 0.01 and ΔPS =  ± 0.1 were indicated as blue and green dashed lines, respectively. D Limited overlapping between flg22-DEGs and flg22-DASs. The Venn diagram between flg22-DEGs (orange circle) and flg22-DASs (pink circle) in WT plants shows the percentages and corresponding gene numbers indicated in each group. E Gene ontology (GO) analysis of flg22-DASs in WT. The statistically enriched gene ontology terms were identified based on the frequency of up-/downregulated flg22-DASs annotated to their frequency in the genome with the cut-off of fold enrichment ≥ 1 and false discovery rate (FDR) < 0.05. F Diagrams of gene structures for three representative flg22-DASs in WT. Solid lines indicate introns, black boxes represent exons, and purple boxes denote alternatively spliced regions. Constitutive, constitutive splicing isoform; IR, intron retention. G Diagrams of protein domains for three representative flg22-DASs in WT. Proteins encoded by constitutive and splicing variant transcripts are designated as β and α forms, respectively. Distinct functional domains with various colored boxes were labeled in the figure. H Relative isoform abundances of three representative flg22-DASs in WT. The isoform usage (IU) was calculated by the percentage abundance of a transcript compared to the total expression of the gene. The blue line represents constitutive splicing transcript (β form), which was defined by a major isoform containing all exons among all splicing variants, and the yellow line represents alternative splicing transcripts (α form), respectively. The expression levels of individual transcripts were retrieved from RNA-seq data. I RT-qPCR analysis of individual splicing variants from three representative flg22-DAS genes. Two-week-old seedlings were treated with or without 100 nM flg22 for 60 min for RT-qPCR analysis with primers specific to each splicing variant. Relative expressions of target transcripts were normalized to UBQ10, and data are shown with mean ± S.D. from three biological repeats (n = 3). Data were analyzed by unpaired two-tailed Student’s t-test between mock- and flg22-treatment. Non-statistically (ns) and statistically significant differences with the corresponding p values were indicated in the figure