Genome-wide alternative splicing landscapes modulated by biotrophic sugarcane smut pathogen

Abstract

Alternative splicing (AS) promotes transcriptome and proteome diversity during growth, development, and stress responses in eukaryotes. Genome-wide studies of AS in sugarcane (Saccharum spp.) are lacking, mainly due to the absence of a high-quality sequenced reference genome, sugarcane's large, complex genome, and the variable chromosome numbers and polyploidy of sugarcane cultivars. Here, we analyzed changes in the sugarcane isoform-level transcriptome and AS landscape during infection with the smut fungus (Sporisorium scitamineum) using a hybrid approach involving Sorghum bicolor reference-based and Trinity de novo mapping tools. In total, this analysis detected 16,039 and 15,379 transcripts (≥2 FPKM) at 5 and 200 days after infection, respectively. A conservative estimate of isoform-level expression suggested that approximately 5,000 (14%) sugarcane genes undergo AS. Differential expression analysis of the alternatively spliced genes in healthy and smut-infected sugarcane revealed 896 AS events modulated at different stages of infection. Gene family and gene ontology functional enrichment analysis of the differentially spliced genes revealed overrepresentation of functional categories related to the cell wall, defense, and redox homeostasis pathways. Our study provides novel insight into the AS landscape of sugarcane during smut disease interactions.

Figure 1

Experimental design and data analysis flowchart. Samples were collected at 5 and 200 DAI and subjected to RNA-seq analysis using three biological replicates for each sample. Twelve libraries were sequenced representing control and stress conditions at the two time points. Data were analyzed using a hybrid approach comprising reference genome (Sorghum bicolor)- and de novo (Trinity)-based mapping and assembly to identify alternative transcripts and isoform-level regulation.

Figure 2

Global distribution of RNA-seq reads along Sorghum bicolor chromosomes. RNA-seq read density (log₁₀ of absolute read count per gene) of the four sugarcane samples (5 DAI control, 5 DAI stress, 200 DAI control, and 200 DAI stress) across the 10 Sorghum bicolor chromosomes. Red dots indicate centromeric regions.

Figure 3

Isoform-level expression analysis in 5 and 200 DAI samples. (A,B) Volcano plots of differentially expressed isoforms at 5 and 200 DAI, respectively. Green and red dots indicate isoforms significantly upregulated (log₂ ≥ 1 and P < 0.05) and downregulated (log₂ ≤ -1 and P < 0.05), respectively, during smut infection. Hierarchical clustering and isoform-level expression dynamics of putative genes associated with (C) cell wall modification, (D) defense signaling, (E) transcription factors, and (F) ROS scavenging are represented by heatmaps (blue: upregulated isoforms; yellow: downregulated isoforms). List of the isoforms used in heatmap analysis are provided in Supplementary Data Set 2.

Figure 4

Frequency of AS events identified in RNA-seq data. Distribution of intron retention (IR), exon skipping (ES), alternative donor (AD), and alternative acceptor (AA) AS events under 5 and 200 DAI control (C) and infected (I) conditions.

Figure 5

GO and gene family (GenFam) enrichment analysis of induced isoforms in response to smut infection at 200 DAI. (A) Enriched GO terms visualized using Cytoscape (see Materials and Methods). GO term interaction networks based on ancestor–child relationships for ‘biological process,’ ‘molecular function,’ and ‘cellular component’ categories. Node size indicates the number of genes in that GO term, and the color scale corresponds to Benjamini-Hochberg P-values. (B) Gene family enrichment analysis of induced isoforms in response to smut infection at 200 DAI using the GenFam tool.

Figure 6

Gene isoform switching at 200 DAI in response to smut infection. Isoform expression changes in genes encoding A) xyloglucan endotransglucosylase/hydrolase (XTH) and D) lipoxygenase (LOX2) (200 C: 200 DAI Control; 200 S: 200 DAI infected). Isoform structures of B) XTH and E) LOX2 with known and novel isoforms, and their corresponding protein structures (C and E). Untranslated and intronic regions are represented by dotted and solid lines, respectively. Left- and right-facing arrows indicate transcript strand orientation.

Figure 7

RT-PCR analysis of differentially expressed sugarcane gene isoforms. Alternative splicing in selected sugarcane genes was analyzed using RT-PCR. Molecular size (bp) of the isoforms is shown using a DNA ladder. Putative alternative transcripts/isoforms are labeled numerically. −: negative control/no template; *: primer-dimer. The top and bottom panels were cropped and grouped from different parts of the same gel for clarity. An uncropped image used to prepare this figure is presented in Supplementary Fig. S3.