Transcriptomic analysis of wheat leaves in response to S. graminum at different time points
Global transcriptomic changes in response to phytotoxic aphid feeding were examined in leaves of wheat seedlings infested with S. graminum at 2, 6, 12, 24 and 48 hpi. A total of 166.41 Gb of clean data were obtained from the eighteen leaf samples, and each of these samples contained ≥7.6 Gb of data with Q30 quality scores ≥ 92.32% (Table S1). Subsequently, 84.6% to 87.92% of the clean reads from each sample were aligned onto the wheat reference genome and matched to either unique or multiple genomic locations (Table S2).
The gene expression levels of genes were used to conduct a PCA for each of the biological replicates. Each replicate from the same group was clustered closely together, which suggested that the repeatability of each treatment was satisfactory, and the samples from different time points of S. graminum infestation were clustered far from each other and the control groups, which indicated that aphid feeding induced significant changes in gene expression (Figure 1A).
The P value≤0.01 (false discovery rate [FDR] adjusted) and log2-fold change (Log2FC) ≥1 or ≤−1 were set as thresholds for DEGs in wheat leaves at different time points. Then, these identified DEGs were used for further analysis. A total of 125,289 DEGs were identified in wheat leaves at different time points (2, 6, 12, 24 and 48 hpi) of aphid feeding (Table S3). Briefly, 9,761 (9,105 up- and 656 downregulated), 22,183 (13,935 up- and 8,248 downregulated), 29,875 (16,214 up- and 13,661 downregulated), 32,741 (17,771 up- and 14,970 downregulated) and 30,729 (17,523 up- and 13,206 downregulated) DEGs were identified at 2, 6, 12, 24 and 48 hpi, respectively (Figure 1B). The distribution of up- and downregulated genes was calculated for each time point and are presented in a Venn diagram (Figure 1C and 1D). Although a unique set of genes increased at each time point (total 74,548), the expression levels of a large number of genes (5,800) were significantly upregulated at all time points. In addition, a unique set of genes was significantly downregulated at each time point (total 50,741), and only 349 genes showed decreased expression at all five time points.
Gene Ontology (GO) analysis of DEGs
GO analysis was used for the functional classification of the DEGs in wheat leaves after aphid infestation. The top 30 enriched GO terms of all DEGs are shown in Figure S1. GO analysis of DEGs induced by S. graminum feeding at early time points is shown in Figure 2. At 2 hpi (Figure 2A, 2B), within the biological process category, the upregulated DEGs were mainly enriched in metabolic process, single-organism process and phosphorus metabolic process. Within the molecular function category, the largest proportion of upregulated DEGs induced by the aphid feeding was enriched in catalytic activity and transferase activity. At 6 and 12 hpi (Figure 2C-2F), the majority of the upregulated DEGs activated by the aphid feeding were enriched in metabolic processes and single-organism processes within the biological process category, protein kinase activity and phosphotransferase activity. In the molecular function category, the upregulated DEGs were mainly enriched in catalytic activity and transferase activity.
At 6 and 12 hpi (Figure 2C-2F), within the biological process category, the downregulated DEGs were mainly enriched in protein photosynthesis and photosynthesis, light reaction. In the cellular component group, greater percentages of DEGs in the cellular component category were enriched in cell and cell parts. In addition, many downregulated DEGs at 6 and 12 hpi were also enriched in chloroplasts, further indicating the damage in wheat leaves caused by S. graminum feeding.
Chlorophyll content in wheat leaves after S. graminum feeding
Transcriptome analysis showed that aphid feeding negatively affected the photosynthetic processes of wheat, and the transcript levels of many light-harvesting- and photosystem-associated genes, such as ribulose-1,5-bisphosphate carboxylase, chlorophyll a-b binding proteins, ferredoxin thioredoxin reductase, and PsbP family proteins, were significantly downregulated (Table 1).
The results in Figure 3 suggested that the total chlorophyll content in wheat leaves at 2, 6 and 12 hpi was not significantly different from that of the control. However, the total chlorophyll content was significantly decreased to 1.49 ± 0.10 mg g−1 FW after 24 h of aphid feeding (F5,18 = 9.447, P=0.0001) and was further reduced to 1.07 ± 0.11 mg g−1 FW at 48 hpi, which was significantly lower than that of the control (2.58 ± 0.18 mg g−1 FW).
Transcript levels of genes involved in SA- and JA-dependent defence pathways in wheat leaves after S. graminum feeding
Phytohormone metabolic pathways are commonly used by plants for defence against both pests and pathogens. The transcriptome data in Table 2 showed that all six phenylalanine ammonia-lyase (PAL) genes involved in SA biosynthesis were significantly upregulated in response to S. graminum at different time points, and the expression levels of PAL gradually decreased with increased aphid feeding time (4.96 to 16.16-fold). Furthermore, PR genes that respond to SA were also significantly upregulated during all time points of aphid feeding (4.92 to 20.59-fold).
A greater effect on genes involved in JA metabolism was observed over time (Table 2). Three lipoxygenase (LOX) genes were significantly upregulated by S. graminum feeding at different time points (1.49 to 9.25-fold), and one lipoxygenase (LOX) was only upregulated at 2 hpi (5.89-fold). The expression levels of allene oxide cyclase (AOC) were also significantly increased at various aphid feeding time points. In contrast, the jasmonic acid-amido synthetases (JARs) were downregulated in infested plants (-2.31 to -3.41-fold). There were also five fatty acid desaturase (FAD) genes that had variable expression levels, with the majority being upregulated (1.36 to 6.77-fold) during infestation. However, some were also downregulated during the three later time points (-1.20 to -2.45-fold), which suggests that the expression of FAD genes may be fine-tuned during defence responses.
The mitogen-activated protein kinase (MAPK) cascade is a key signalling pathway of plant defence, and WRKY transcription factors (TFs) appear to be regulated by MAPKs and involved in the regulation of plant defence. The transcript levels of several MAPKs were significantly upregulated (1.01- to 3.48-fold) in response to S. graminum feeding at 12, 24 and 48 hpi. Additionally, several WRKY TFs in wheat leaves were significantly induced (1.59 to 11.14-fold) in response to aphid feeding at different time points (Table 3).
Effects of S. graminum feeding on hydrogen peroxide (H2O2) accumulation and the activity of antioxidant enzymes in wheat leaves
As shown in Table 4, S. graminum feeding upregulated various ROS-scavenging genes, such as POD and SOD (superoxide dismutase), at 2 hpi. The expression levels of these two genes were increased at 6, 12 and 24 hpi and then gradually decreased at 48 hpi, but the transcript levels were still significantly increased compared with the control levels. The expression levels of catalase (CAT) genes showed no significant differences at 2 hpi compared to the control levels. Among them, two genes were significantly downregulated at 6, 12, 24 and 48 hpi, and the other two genes were significantly upregulated.
The activities of three antioxidant enzymes, POD, SOD and CAT, were also examined in wheat leaves infested with aphids (Figure 4). Compared with the control, the activity of POD was significantly increased after 6 h (19.01±3.94 U mg-1 protein) of S. graminum feeding and reached a peak at 48 hpi (44.44±3.37 U mg-1 protein;F5, 12=10.17, P=0.001). Furthermore, the activities of SOD (55.10±7.55 U mg-1 protein; F5,12=5.15, P=0.009) and CAT (0.20±0.038 U mg-1 protein; F5,12=7.27, P=0.002;) were significantly upregulated after 12 h of S. graminum feeding. The increased activity of ROS scavengers induced by aphid feeding suggested that S. graminum feeding induces oxidative stress in wheat leaves.
To further examine the effects of aphid feeding on oxidative stress in wheat, S. graminum-infested leaves were examined after cytological staining with 3,3’-diaminobenzidine (DAB), which was used to detect the production of H2O2. As shown in Figure 5, small and obvious brown spots were detected at 2 hpi, indicating H2O2 accumulation at the aphid feeding site. The number and size of the spots increased with increasing time aphid feeding time.
Effects of inhibition of NADPH oxidases on H2O2 accumulation and defence responses in wheat leaves
To detect the roles of plasma membrane NADPH oxidases in H2O2 accumulation induced by S. graminum feeding, wheat leaves were treated with the NADPH oxidase inhibitor DPI. The DAB staining results, shown in Figure 6A, indicate that many obvious brown spots were detected at the aphid feeding sites of infested leaves, but fewer brown spots were observed after 10 μM and 25 μM DPI treatments, indicating that the H2O2 production induced by aphid feeding was inhibited by DPI. Additionally, the H2O2 contents were significantly decreased to 50.60 ± 9.51 μmol g-1 FW and 33.93 ± 3.00 μmol g-1 FW in wheat leaves treated with 10 μM and 25 μM DPI, respectively (F2,6 =6.44, P=0.032) (Figure 6B).
As shown in Figure 7A and 7B, DPI treatment had significant effects on the expression levels of defence response genes in wheat leaves. The expression levels of the salicylic acid-related genes PAL and PR1 were significantly reduced in DPI-treated wheat leaves compared to the control (wheat leaves infested with aphids for 24 h) (F2,6=10.96, P=0.01; F2,6=65.53, P<0.001).