RNA-Seq analysis of a soybean near-isogenic line carrying bacterial leaf pustule-resistant and -susceptible alleles

Abstract

Bacterial leaf pustule (BLP) disease is caused by Xanthomonas axonopodis pv. glycines (Xag). To investigate the plant basal defence mechanisms induced in response to Xag, differential gene expression in near-isogenic lines (NILs) of BLP-susceptible and BLP-resistant soybean was analysed by RNA-Seq. Of a total of 46 367 genes that were mapped to soybean genome reference sequences, 1978 and 783 genes were found to be up- and down-regulated, respectively, in the BLP-resistant NIL relative to the BLP-susceptible NIL at 0, 6, and 12h after inoculation (hai). Clustering analysis revealed that these genes could be grouped into 10 clusters with different expression patterns. Functional annotation based on gene ontology (GO) categories was carried out. Among the putative soybean defence response genes identified (GO:0006952), 134 exhibited significant differences in expression between the BLP-resistant and -susceptible NILs. In particular, pathogen-associated molecular pattern (PAMP) and damage-associated molecular pattern (DAMP) receptors and the genes induced by these receptors were highly expressed at 0 hai in the BLP-resistant NIL. Additionally, pathogenesis-related (PR)-1 and -14 were highly expressed at 0 hai, and PR-3, -6, and -12 were highly expressed at 12 hai. There were also significant differences in the expression of the core JA-signalling components MYC2 and JASMONATE ZIM-motif. These results indicate that powerful basal defence mechanisms involved in the recognition of PAMPs or DAMPs and a high level of accumulation of defence-related gene products may contribute to BLP resistance in soybean.

Figure 1.

Disease symptoms in BLP-susceptible and BLP-resistant NILs after Xag inoculation. Small yellow to brown lesions with a raised pustule typically formed in the early disease stage, with large necrotic lesions developing later.

Figure 2.

Number of gene transcripts in the BLP-resistant NIL that were up- and down-regulated [P < 0.001 and log₂ (fold change) >2.0 or<−2.0] compared with the BLP-susceptible NIL. The number inside the parentheses indicates the number of genes expressed at the hours after Xag inoculation (hai). The total number of gene transcripts is at the bottom of each Venn diagram.

Figure 3.

Cluster analysis of 2415 genes differentially expressed following Xag inoculation. The genes were classified based on similarity of expression pattern over the time course of infection. Ten clusters were identified by K-means clustering. The pink lines indicate representative transcriptional regulators; x- and y-axes represent hours after Xag inoculation (hai) and log₂ fold change [log₂ (BLP-resistant NIL/BLP-susceptible NIL)], respectively.

Figure 4.

Schematic diagram of plant immunity to bacterial pathogens, adapted from Dodds and Rathjen. Plants use two strategies to respond to pathogen attacks: PTI and ETI. Ultimately, through several branched and multi-component pathways, defence-related genes are transcribed. Genes that were up-regulated in the BLP-resistant NIL are represented as reddish bold characters.

Figure 5.

Heat maps of gene transcripts in BLP-susceptible and BLP-resistant NILs after Xag infection [P < 0.001 and log₂ (fold change) >2.0 or<−2.0]. PTI-, ETI-, JA-, and defence-related soybean genes whose expression differed significantly are represented. Darker colours indicate higher transcript levels.

Figure 6.

Validation of RNA-Seq data by qRT–PCR. Nine PTI-, ETI-, and defence-related genes were selected for validation.