Strand-Specific RNA-Seq Analyses of Fruiting Body Development in <i>Coprinopsis cinerea</i>

<div><p>The basidiomycete fungus <i>Coprinopsis cinerea</i> is an important model system for multicellular development. Fruiting bodies of <i>C</i>. <i>cinerea</i> are typical mushrooms, which can be produced synchronously on defined media in the laboratory. To investigate the transcriptome in detail during fruiting body development, high-throughput sequencing (RNA-seq) was performed using cDNA libraries strand-specifically constructed from 13 points (stages/tissues) with two biological replicates. The reads were aligned to 14,245 predicted transcripts, and counted for forward and reverse transcripts. Differentially expressed genes (DEGs) between two adjacent points and between vegetative mycelium and each point were detected by Tag Count Comparison (TCC). To validate RNA-seq data, expression levels of selected genes were compared using RPKM values in RNA-seq data and qRT-PCR data, and DEGs detected in microarray data were examined in MA plots of RNA-seq data by TCC. We discuss events deduced from GO analysis of DEGs. In addition, we uncovered both transcription factor candidates and antisense transcripts that are likely to be involved in developmental regulation for fruiting.</p></div

GO analysis of DEGs in each transition.

<p>GO analysis of DEGs in each transition.</p

Schematic diagram of fruiting body development in <i>C</i>. <i>cinerea</i>.

<p>The 13 stages/tissues (numbers in circles) were selected to investigate the transcriptome by RNA-seq. Developmental and cellular events in each stage/tissue are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141586#pone.0141586.t001" target="_blank">Table 1</a>.</p

A model for significant developmental transitions based on GO analysis.

<p>Notable events among top 3 categories are depicted in fruiting. Events detected by up-regulated DEGs and down-regulated DEGs are indicated in the upper part with upward red arrows and in the lower part with downward blue arrows, respectively. The asterisks and bidirectional light green arrows indicate that similar annotation terms are enriched in both up-regulated and down-regulated DEGs.</p

Samples used for RNA-seq.

<p>Samples used for RNA-seq.</p

Transcription factor candidates in <i>C</i>. <i>cinerea</i> and changes in the expression levels.

<p>(A) Relative distribution of Pfam domains in the TFCs. A total of 848 TFCs were sorted on the basis of Pfam domains, and the number and percentage of each Pfam are indicated. (B) A histogram dividing 848 transcription factor candidates into bins based on their RPKM values. Red line shows a total number of the TFC genes, 848. Red arrows indicate notable changes.</p

Changes in gene expression of TFCs with the same Pfam domain.

<p>(A) Top 4 genes of C2H2 transcription factors. (B) Top 4 genes of fungal Zn(2)-Cys(6) binuclear cluster domain. (C) Top 4 genes of zinc finger, C3HC4 type (RING finger). (D) Top 4 genes of HMG (high mobility group) box. (E) Top 4 genes of fungal specific transcription factor domain. (F) Top 4 genes of homeobox domain.</p

Hyphal knot induction.

<p>(A) The mycelium was cultured in the 12 hr light/ 12 hr dark cycle. (B) The mycelium was cultured in dark for 5 days, followed by 2 hr light and 24 hr dark. The hyphal knots were induced at the area indicated by parenthesis. Red dots indicate the position of apical hyphae when light was received.</p

Clustering of samples using RPKM values of sense transcripts.

<p>Hierarchical clustering of samples was performed using RPKM values of sense transcripts from all gene models. The trees were depicted with MeV [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141586#pone.0141586.ref063" target="_blank">63</a>] by using average linkage distance measurement and Pearson’s correlation (A) and Spearman’s correlation (B). The vertical scale is correlation coefficient. Red dots indicate separated 2_Knot samples. Green, blue and yellow dots also indicate duplicate samples that are not located in a single clade.</p

Antisense transcripts.

<p>(A) Change in the percentages of the antisense reads. (B) Antisense reads in Protein Id: 13022 (CC1G_15471). Left panel: Change in sense and antisense RPKM values. Red arrowheads indicate the points compared in the right panel. Right panel: R2 reads were mapped to the genomic sequences and separated based on read strands in IGV. (C) Antisense reads in Protein Id: 4305 (CC1G_04978), as shown in (B). Mapping of R1 reads showed similar patterns to those of R2 reads shown.</p