https://orcid.org/0000-0003-3533-1363
Figures & data
Table 1. Statistics of KJ201 genome
Table 2. Statistics of KJ201 transcripts analysed
Figure 1. Characteristics of AS in M. oryzae KJ201 under different conditions.
(a) Among 13,306 annotated genes of KJ201, 2,413 are subjected to AS under one or more conditions. Transcript isoforms from 29 and 499 genes were detected only in mycelia and during infection, respectively. The remaining 1,883 genes produced AS transcripts under both conditions. (b) The number of AS genes in each sample predicted using three FPKM values for cut-off. (c) PCA analysis of the annotated transcripts and their isoforms. The log2 FPKM values of individual transcripts within each sample are used to calculate PCA distances. The standard PCA function implemented in prcomp was used for this analysis.
Figure 2. AS repertoires and the expression patterns of putative AS regulator genes under different conditions.
(a) Venn diagram showing the number of AS isoforms and genes (in parenthesis) at each stage. The light grey dashed region denotes the AS isoforms produced in mycelia as well as during infection (2,095). Each stage is colour-coded: mycelia (grey line), pre-penetration (yellow line), biotrophic (red and green line), and necrotrophic (blue and purple line). The stage-specific isoforms are colour coded. The AS isoforms detected only during infection are shown in a dashed red line. (b) Heatmap showing how the SR and hnRNP protein-coding genes are expressed under these conditions.
Figure 3. Three types of AS isoforms produced under different conditions and characteristics of Switching AS-type isoforms.
(a) The diagrams show the relative abundance (the ratio of isoforms over annotated forms) of three types of AS transcripts during the growth stages analysed. Constitutive AS and Low-frequency AS isoforms indicate those with the relative abundances of > 0.5 and < 0.5, respectively. Switching AS-type isoforms include those fluctuating between the first two types between mycelia and infected rice. (b) Expression patterns of the following four groups of Switching AS-type isoforms during infection: grey box (the isoforms abundantly produced at all infection stages), yellow box (those produced higher than corresponding annotated forms only at the pre-penetration stage, 18 hpi), green box (those produced higher than corresponding annotated forms only at the biotrophic stage, 25 hpi or 36 hpi), and orange box (those produced higher than corresponding annotated forms only at the necrotrophic stage, 45 hpi or 72 hpi). (c) The GO terms enriched among the genes that produce Switching AS-type isoforms (P-value < 0.05).
Figure 4. Four types of AS observed and their distribution patterns under different conditions.
(a) Four types of AS, including intron retention (IR), exon skipping (ES), alternative 3’ splicing site (A3SS), and Alternative 5’ splicing site (A5SS), observed at the vegetative and five infection stages are shown. The black box denotes exon. (b) Sequence patterns at the splicing sites in annotated genes (left) and isoforms (right). The LOGO diagrams show 5 bp downstream and upstream from the splicing junctions.
Figure 5. Domain variation patterns observed among the translated isoforms.
(a) A filtration pipeline used to remove non-translated isoforms and isoforms with low coding potential. (b) Proportions of NMD candidates (PTC+) in four major isoform types: Intron retention (IR), ES (Exon skipping), A3SS (Alternative 3’ splicing site), and A5SS (Alternative 5’ splicing site) (c) The pie graphs show the distribution patterns of domain change patterns among translated AS transcripts.
Figure 6. Predicted modifications of secreted proteins due to AS.
(A) Three types of changes that potentially affect protein secretion are shown. Two types, loss of signal peptide (SP) and gain of transmembrane motifs (TM), are expected to disrupt protein secretion. A gain of SP was also observed. (B) The expression pattern of AS isoforms of secreted protein-coding genes.
Table 3. List of the proteins produced using transcript isoforms generated via AS
Figure 7. Validation of the transcripts produced via AS and the resulting structural changes.
AS of selected genes was validated using RT-PCR. The samples analysed are mycelia and rice leaves collected at 3 days post-inoculation (dpi) and 6 dpi. Genomic DNA was used as the control for RT-PCR reactions. The structural differences between the annotated form (grey) and isoform (blue) are shown. The red boxes indicate the amplified regions of DNA and cDNA.
Supplemental material
