High-Throughput RNA Sequencing of Pseudomonas-Infected Arabidopsis Reveals Hidden Transcriptome Complexity and Novel Splice Variants

We report the results of a genome-wide analysis of transcription in Arabidopsis thaliana after treatment with Pseudomonas syringae pathovar tomato. Our time course RNA-Seq experiment uses over 500 million read pairs to provide a detailed characterization of the response to infection in both susceptible and resistant hosts. The set of observed differentially expressed genes is consistent with previous studies, confirming and extending existing findings about genes likely to play an important role in the defense response to Pseudomonas syringae. The high coverage of the Arabidopsis transcriptome resulted in the discovery of a surprisingly large number of alternative splicing (AS) events – more than 44% of multi-exon genes showed evidence for novel AS in at least one of the probed conditions. This demonstrates that the Arabidopsis transcriptome annotation is still highly incomplete, and that AS events are more abundant than expected. To further refine our predictions, we identified genes with statistically significant changes in the ratios of alternative isoforms between treatments. This set includes several genes previously known to be alternatively spliced or expressed during the defense response, and it may serve as a pool of candidate genes for regulated alternative splicing with possible biological relevance for the defense response against invasive pathogens

Differentially Expressed Genes (DEG).

<p>A) By treatment comparison at 1 hpi, B) by treatment comparison at 6 hpi, C) by treatment comparison at 12 hpi, and D) all treatment comparisons, by time point.</p

Novel intron retention event in gene At4g16890.

<p>This gene “encodes a Toll Interleukin1 receptor-nucleotide binding-Leucine rich repeat-type resistance gene (TIR-NB-LRR-type) involved in the salicylic acid-dependent defense response pathway. Mutant plants constitutively express pathogenesis-related (PR) genes and are pathogen resistant. Resistance signaling in snc1 requires EDS1, MOS3 and PAD4”. In this case, the event is described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074183#pone.0074183-Xu1" target="_blank">[34]</a> and corresponds to a TAIR 10 “B-List” gene.</p

Differentially Expressed Isoforms (DEI).

<p>A) By treatment comparison at 1 hpi, B) by treatment comparison at 6 hpi, C) by treatment comparison at 12 hpi, and D) all treatment comparisons, by time point.</p

Differentially alternatively spliced (DEI + DIR) isoforms.

<p>A) By treatment comparison at 1 hpi, B) by treatment comparison at 6 hpi, C) by treatment comparison at 12 hpi, and D) all treatment comparisons, by time point.</p

Novel cassette exon event in gene At5g45190 “Cyclin T partner CYCT1;5”.

<p>Putative cassette exon(s) are indicated by the red arrow. This gene, which has been previously shown to play an important role in infection with Cauliflower mosaic virus <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0074183#pone.0074183-Cui1" target="_blank">[29]</a>, has a TAIR 10 B-List transcript which confirms the indicated exon skipping event.</p

Major isoform percentages.

<p>Distribution of the IQ.OWLS estimates for the major (most highly expressed) isoform in 2-isoform genes expressed with at least 500 reads (1,695 out of 4,318 genes) in mock treated leaves at 6 hpi.</p

Distribution of gene expression.

<p>Shown is a histogram of the mean log₂ IQ.OWLS FPKM expression levels for the 33,602 Arabidopsis genes in TAIR 10.</p

Summary of detected AS events.

<p>Summary of detected AS events.</p