Transcriptome Analysis Identifies a 140 kb Region of Chromosome 3B Containing Genes Specific to Fusarium Head Blight Resistance in Wheat

Abstract

Fusarium head blight (FHB), mainly caused by Fusarium graminearum, is one of the most destructive fungal diseases of wheat (Triticum aestivum L.). Because of the quantitative nature of FHB resistance, its mechanism is poorly understood. We conducted a comparative transcriptome analysis to identify genes that are differentially expressed in FHB-resistant and FHB-susceptible wheat lines grown under field conditions for various periods after F. graminearum infection and determined the chromosomal distribution of the differentially expressed genes (DEGs). For each line, the expression in the spike (which exhibits symptoms in the infected plants) was compared with that in the flag leaves (which do not exhibit symptoms in the infected plants). We identified an island of 53 constitutive DEGs in a 140 kb region with high homology to the FhbL693b region on chromosome 3B. Of these genes, 13 were assigned to specific chloroplast-related pathways. Furthermore, one gene encoded inositol monophosphate (IMPa) and two genes encoded ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Our findings suggest that the temporary susceptibility in locally infected spikes results from the cross-talk between RuBisCO and IMPa, which blocks secondary signaling pathways mediated by salicylic acid and induces a systemic acquired resistance in the distant leaf tissue.

Keywords: fusarium head blight; gene island; photosynthesis; transcriptome; wheat.

Figure 1

Comparisons of L693 and L661 in terms of Fusarium head blight (FHB) severity and differentially expressed genes. (A) Photograph of spikes of L693 (left) and L661 (right), grown under greenhouse conditions, 21 days after inoculation with Fusarium graminearum in 2013. For L693, disease symptoms are visible only in the inoculated spikelet, whereas, for L661, they are evident in the entire spike. Bars, 1 cm. (B) Comparison of kernel health between the resistant line L693 (left) and the susceptible line L661 (right) in 2013. The kernels of L693 were fully filled, while those of L661 were shriveled and pinkish. Bars, 1 cm. (C) Multiple comparison of the percentage of diseased spikelets (PDS) in 2013 and 2014. Error bars indicate the standard error of PDS;. Means with the same letter (above error bar) are not significantly different (p > 0.01) and with different letter are significantly different (p ≤ 0.01).

Figure 2

Comparison of differentially expressed genes in L693 and L661 following FHB infection. Number of upregulated genes (left, yellow circle), downregulated genes (right, blue circle), and genes without differential expression (middle, purple circle) in L693 (resistant) compared to L661 (susceptible) at 0, 24, and 72 h after inoculation (hai) in spike (A) and leaf (B).

Figure 3

The reliability of RNA-seq data, as demonstrated by qRT-PCR (Quantitative real time polymerase chain reaction) and sample clustering. (A) Correlation between normalized mRNA-seq RPKM results and qRT-PCR expression values. The scatterplot shows the log₂ fold change of RPKM and qRT-PCR expression values; a trend line is shown in red; (B) sample clustering based on counts of mapped Illumina reads. The dendrogram represents the hierarchical clustering of samples as determined by Euclidean distance. The heat map shows a false-color representation of the sample correlation value.

Figure 4

Log-fold change (logFC) against average logRPKM in different genotypes. (A–F) Differentially expressed genes with a false discovery rate (FDR) of less than 5% and a log-fold change larger than 2 are highlighted in red. In each panel, the red dots above the upper blue line (logFC > +2) represent the upregulated genes in L693 or the downregulated genes in L661; the red dots blow the lower blue line (logFC < −2) represent the upregulated genes in L661 or the downregulated genes in L693. The blue dots represent gene(s) with extreme values of −12 > logFC > 12 or with an average logRPKM > 3. The green symbols represent genes of interest in this study; rectangle for MSTRG.24516, circle for MSTRG.24551, triangle for MSTRG.24552.

Figure 5

Venn diagram showing the number of differentially expressed genes shared by L693 and L661 at 0 h (red), 24 h (green), and 72 h (blue) after inoculation for the spike (A) and leaf (B). The numbers without parentheses represent the differentially expressed genes across the whole genome; the numbers in parentheses represent the differentially expressed genes on wheat chromosome 3B.

Figure 6

Distribution of genes on wheat chromosome 3B. (A) Eighty-six genes annotated by the Genetics Diversity Ecophysiology of Cereals (GDEC) group at the French National Institute of Agronomic Research (INRA) located in region 181.40–181.54 Mb of wheat chromosome 3B. (B) Fifty-three expressed genes located in region 181.40–181.54 of chromosome 3B in this study. The insets show enlargements of the boxed regions.

Figure 7

Distribution of gene expression levels on chromosome 3B in all 12 samples. The x-axis represents the chromosome range 0–774.43 Mb. The y-axis represents the normalized log (RPKM+1) range 0–1. The region included in the dashed rectangle indicates the 181.40–181.54 Mb region. Distribution of gene expression level on chromosome 3B for six spike samples (A) and six leaf samples (B). Distribution of gene expression level in the gene island at region 181.40–181.54 Mb of chromosome 3B for six spike samples (C) and six leaf samples (D).

Figure 8

A proposed working model of the roles of MSTRG.24516, MSTRG.24551, and MSTRG.24552 in the regulation of FHB resistance in wheat line L693. (A) In the early infection stage, FHB induces temporary susceptibility locally in a tissue; (B) this then leads to system-acquired resistance in distal tissues.