Global transcriptome changes in grape during E. ampelina infection
A total of 3414 DEGs were found using DESeq analysis from the HR and HS grapes in at least one time point, among which 2,246 were detected in the HR grape samples and 2,019 DEGs in the HS grape samples (Fig. 1a, Additional file 2: Table S2). To identify E. ampelina-responsed genes, we compared the numbers of DEGs to their control at the same time point. Figure 1 showed that the numbers of up-regulated genes were consistently higher than down-regulate in either HR or HS cultivars, except for the HR48 (HR at 48 hpi) sample.
Venn diagram analysis of the DEGs among the five HR time points revealed limited overlap (24 DEGs) across all five time points, but a much greater overlap (427 sequences) between the HR12 and HR24 time points (Fig. 2a). Similarly, we identified 32 genes in common between all five HS time points and 132 between the HS12 and HS24 samples (Fig. 2b). The greatest number of DEGs were distinctive for specific datasets (i.e. 722 in HR12, 1,328 in HR24, 711 in HS6, and 945 in HS72 compared to the controls) with relatively little overlap (8 DEGs; Fig. 2c) across all the samples. These results suggest that gene expression in HR and HS is different with specific genes up- or down-regulated depending on the infection period. We next analyzed the expression patterns of these common DEGs using Genesis software , and found that the 24 common DEGs identified from the HR grape, were all up-regulated throughout the duration of the experiment, except for a NAC transcription factor, which was down-regulated at the late stage of infection (Fig. 2a). Similarly, the 32 common DEGs identified from the HS samples, the majority of which were transcription factors, were also induced at all time points, except one glycine rich protein transcript, which was down-regulated at 48 h (Fig. 2b). Our data indicated that there was little difference in the identity and number of common DEGs between HR and HS grapes. Furthermore, the eight common DEGs identified from both HR and HS grapes showed significant up-regulation during all the time points in both species (Fig. 2c), suggesting a potential role in grape defense against E. ampelina, although the biological functions of these genes are still unknown.
Genesis K-means analysis of the 3414 DEGs revealed four major expression pattern groups (Fig. 3a). Expression profiles of most genes in group I (390 DEGs) were relatively stable during all the five time points in the HS grape, but up- and/or down-regulated significantly in the HR grape from 6 to 24 hpi. Based on this expression variety in the HR grape, we divided group I DEGs into three sub- clusters. Majority of the 188 DEGs in cluster 1 showed stable expression 6–12 hpi, then were highly induced at 12–24 hpi. Cluster 2 genes (165) showed rapid greater expression at 6–12 hpi, while many genes in cluster 3 (37 DEGs) were continuously induced from 6 to 48 hpi in the HR grape. Group II expression (263 DEGs) profiles varied dramatically in both the HR and HS grapes during the whole infection time. These genes could be classified into three sub-clusters. Cluster 4 DEGs (117) showed similar expression profiles in both the two grape species at whole time, except between 6–12 hpi, where they were induced in the HR grape but suppressed in the HS grape. However, the expression profiles of genes in cluster 5 (79 genes) were largely opposite between the two grape species during the whole time. Cluster 6 genes (165) were substantially up-regulated during the initial stage of infection, but down-regulated at 48 hpi in both species. Group III expression (548 DEGs) profiles showed the opposite expression patterns from group I genes. Most were relatively stable in the HR grape after infection but were up- or down-regulated in HS grape. The expression profiles of genes in cluster 7 (62) and cluster 8 (395) varied similarly in the HS grape during the E. ampelina infection, being up-regulated at 6–12 hpi and 24–72 hpi, but down-regulated at 12–24 hpi, although the changes in expression were more rapid for cluster 7 than for cluster 8 genes. Cluster 9 genes (91) were up-regulated to a minor degree at 6–12 hpi, then remained stable at 12–48 hpi followed by significant up-regulation at 48–72 hpi. Group IV genes (2,213) did not show expression differences between the HR and the HS grapes. Within the three group IV sub-clusters, the patterns of those in cluster 10 genes (847) were very similar between the two species, while most genes in cluster 11 (865) only showed similar expression patterns in both species after 12 hpi. Expression of most cluster 12 DEGs (501) were induced at earlier stages (6–24 hpi) in the HR grape then suppressed during the later stages of infection (48–72 hpi). In contrast, these genes showed suppressed expression at 6–48 hpi followed by induction at 48–72 hpi in the HS grape.
To more clearly identify DEGs linked to E. ampelina infection, the heatmap of the 851 common DEGs between two grapes after E. ampelina infection (Fig. 1a) was generated from the RPKM data (Fig. 3b). Base on the heatmap, the 851 common DEGs were divided into six classes (Fig. 3b; Additional file 3: Table S3). Most genes in Class I, V and VI, involved in stress response and metabolic processes such as phenylalanine ammonia-lyase, receptor-like protein kinase, chalcone synthase, heat shock proteins and cytochrome P450 superfamily proteins, were highly abundant and induced in the HR grape at 6, 12 and 24 hpi. Notably, the expression of these DEGs was induced in the HS grape much later at 72 hpi. However, the expression pattern of Class II genes was largely opposite between two grape species at 6 and 12 hpi. Class III (i.e. 60S ribosomal protein, histone) genes first exhibited down-regulation in both the HR and HS grapes and were significantly down-regulated at 24 hpi. Class IV genes (e.g. ETH responsive transcription factor 1, NAC transcription factor and WRKY transcription factor) were highly induced at 6 hpi in the HS grape and at 24 hpi in the HR grape. Overall, most DEG expression profiles present in both grape species were consistently up- or down-regulation, but the response time was much faster in the HR grape (6 hpi and 24 hpi) than in the HS grape (48 hpi and 72 hpi).
Functional classification and pathway enrichment analysis
The 3,414 DEGs related to E. ampelina infection of grape were arranged into 98 functional categories based on the GO (Gene Ontology) analysis (Additional file 4: Table S4). They comprised three major enrichment categories: ‘molecular functions’ (25), ‘biological processes’ (47) and ‘cellular components’ (26). In the ‘molecular function’ category, genes associated with ‘binding’ (12.7%), ‘protein binding’ (12.4%), ‘nucleotide binding’ (9%), ‘DNA binding’ (5.3%), ‘nucleic acid binding’ (0.9%), ‘RNA binding’ (0.9%), ‘carbohydrate binding’ (0.9%), ‘lipid binding’ (0.6%), ‘chromatin binding’ (0.2%), ‘receptor binding’; (0.1%), and ‘oxygen binding’ (0.04%) were classified as binding, which was the most abundant category (Fig. 4a). Other abundant groups were ‘catalytic activity’ (11.1%), ‘transferase activity’ (7.3%), ‘hydrolase activity’ (6.9%), ‘kinase activity’ (4.4%), ‘receptor activity’ (3.2%), ‘transporter activity’ (2.4%), and two transcription-related groups that include ‘transcription factor activity’ (2.2%) and ‘transcription regulator activity’ (1.84%). In the ‘biological processes’ categories, ‘response to stress’ (8.3%), ‘response to abiotic stimulus’ (4.0%), ‘response to endogenous stimulus’ (3.5%), ‘response to biotic stimulus’ (2.6%), and ‘response to external stimulus’ (1.4%) were grouped in the plant host defense category, which involves plant responses to various abiotic and pathogen/insect stresses. Gene functions in ‘metabolic process’ (6.9%), ‘lipid metabolic process’ (2.1%), ‘protein metabolic process’ (2.0%), ‘carbohydrate metabolic process’ (1.9%) and ‘DNA metabolic process’ (1.2%) represented the second most abundant functional gene group (Additional file 4: Table S4). In addition to these two major categories, additional categories identified included ‘biosynthetic process’ (5.2%), ‘transport’ (3.5%), ‘transcription’ (3.4%), ‘signaling transduction’ (3.3%), and two cell structure groups that included ‘cellular process’ (8.4%) and ‘cellular component organization’ (3.1%). In the ‘cellular component’ category, the abundant groups included ‘membrane’, ‘nucleus’, ‘plasma membrane’, and ‘cytoplasm’ (Fig. 4b; Additional file 4: Table S4). We also found that all the 47 GO biological process terms were present in the two different grapes and that the first five most abundant GO terms were the same, including ‘response to stress’, ‘cellular process’, ‘metabolic process’, ‘biosynthetic process’, and ‘response to abiotic stimulus’ (Fig. 4c; Additional file 5: Table S5). However, genes related to ‘metabolic process’, ‘transcription’, and ‘cellular amino acid and derivative metabolic process’ were more abundant in the HS grape than in the HR grape, where ‘cellular process’, ‘cell cycle’, ‘DNA metabolic process’, and ‘cell death’ were more enriched. These results indicate that many biological processes changed in both the resistant and susceptible grapes after E. ampelina infection, most of which are the same, including some defense-related processes.
For further functional categorization, pathway enrichment analyses were performed using Plant MetGenMAP. As a result, all the significant DEGs from both the HR and HS samples were grouped into 22 biochemical pathways (P < 0.05; Fig. 5a). DEGs HR grape were assigned to 10 significantly changed pathways, and 21 in the HS grape (Fig. 5a). Some of them were involved in plant responses to pathogen infection, and have also been reported by other studies. For example, variations in expression of genes involved in plant hormone and flavonoid biosynthesis following Erysiphe necator-infection in V. pseudoreticulata have been previously reported . Major pathways related to plant responses following E. ampelina infection in HR grape included: flavonoid biosynthesis (53), salicylate biosynthesis (21), phenylpropanoid biosynthesis (21), suberin biosynthesis (23), triacylglycerol degradation (20) and jasmonic acid biosynthesis (9) (Fig. 5b). Pathways unique to the E. ampelina-infected HS grape included methanol oxidation to formaldehyde (8), oxidative ethanol degradation III (8), removal of superoxide radicals (9), cyanide degradation (9) and monoterpene biosynthesis (10). Pathways common to the two genotypes included flavonoid biosynthesis, asparagine biosynthesis, salicylate biosynthesis, phenylpropanoid biosynthesis, suberin biosynthesis, and triacylglycerol degradation (Fig. 5a). Most DEG expression patterns from these metabolic pathways were significantly up-regulated (Fig. 5b). For example, the flavonoid biosynthesis (P1) pathway and the relevant DEG transcripts were enriched at all time points in HR grape after E. ampelina infection. The DEGs involved in P1 were also up-regulated in HS grape, but not until 48 hpi. Only a few DEGs involved in the asparagine biosynthesis pathway were down-regulated in both genotypes at 24 hpi and 72 hpi. Interestingly, jasmonic acid biosynthesis was only enriched in HR grape. We also noticed that there was a higher number of enriched pathways in the HR grape than in the HS grape during the initial stage after infection: eight pathways significantly changing at 6 h in HR and only three in HS grape. Moreover, 71 DEGs from significantly changed pathways were observed in HR grape at 24 hpi, which was also the highest number of all-time points. In the HS grape, the number of enriched pathways gradually increased from three at 6 hpi to 13 at 72 hpi, and the number of DEGs changed from nine at 6 hpi to 113 at 72 hpi. Pathway enrichment analysis revealed that transcripts involved in flavonoid biosynthesis and phenylpropanoid biosynthesis play very important roles in the grape responses to E. ampelina infection. The resistant and susceptible grape varieties responded differently as the significantly changed pathways were observed earlier in the HR grape (6 and 24 hpi) than in the HS grape (48 and 72 hpi).
Genes potentially involved in grape resistance to E. ampelina infection
GO functional and pathway enrichment analyses revealed that there were a lot of defense-related genes undergoing transcriptional changes in both the HR and HS grapes after E. ampelina infection. Most of these genes belong to Class I, II, IV, V and VI described in Fig. 3b (Additional file 3: Table S3), including protein kinases, transcription factors, metabolite synthases, and hormone and pathogen-related genes (Fig. 6; Additional file 6: Table S6). Below, the gene families of interest for potential engineering of pathogen resistance are further described.
Protein kinases and protein
In plant pathogen-induced immunity, the roles of kinases have been well defined. In particular, it has been shown that receptor-like kinases (RLKs) trigger initial immune responses..Interestingly, there were many up-regulated RLKs in both the HR and HS grapes after searching our DEGs data. In general, most of them were significantly induced in the HR grape during earlier stages, and only some were up-regulated in HS grape at later stages. Similarly, several calmodulin-binding proteins and pathogenesis related (PR) proteins including beta-1, 3-glucanase, chitinase, thaumatin-like protein and disease resistance protein were also up-regulated in HR grape during the earlier stages (Fig. 6).
Transcription factors are important regulators of both biotic and abiotic stress responses as part of the immune system .
Transcription factors (TFs) are an integral part of the immune system that are vital in regulating plant defense responses. Many TFs were found in our analysis, which include members of the WRKY, MYB, ERF1, NAC domain, zinc finger and bHLH families (Fig. 6). Most of them play critical roles in plant defense against biotic stress in previous reports [7, 31–34], and many were induced by E. ampelina infection in the HR grape at the earlier stages (Fig. 6). In contrast, a number of the WRKY TFs were up-regulated earlier in HS grape than in HR grape and about one third of them only showed differential expression in the HS grape (Fig. 6). The zinc finger-containing TFs were suppressed at earlier time points after infection in the HR grape but up-regulated in the HS grape, and among the 37 MYBs identified, five were down-regulated at 24 hpi but only in the HR grape (Fig. 6). Besides, we also found lots of heat-shock TFs (HSF) described as crucial regulators in phytohormone signaling. Figure 6 showed the detailed expression patterns of these TFs.
Metabolism and oxidative stress
Among the E. ampelina infection-related genes, there were several genes participating in plant metabolism (Fig. 5). Examples of these genes were involved in phenylpropanoid pathway, such as flavonoid biosynthesis and phenylpropanoid biosynthesis (Fig. 5a). Most of them were induced in the HR grape at 6 hpi and/or 24 hpi but not up-regulated until 48 hpi and/or 72 hpi in the HS grape, suggesting that metabolite synthesis-related genes may also take part in the grape defense against E. ampelina (Fig. 5b).
Reactive oxygen species (ROS) are important in plant defense against biotic stress because they can protect host cellular components from oxidative damaged caused by free radicals released during the oxidative burst .As one of the major ROS scavengers, glutathione is known to play a crucial role in plant response to pathogen infection . We found an enzyme gene involved in glutathione synthesis (glutathione S-transferase; GSTU) was highly induced at earlier time points (6–24 hpi) in the HR grape and later (48–72 hpi) in the HS grape (Fig. 6). Other ROS-related genes including cinnamyl alcohol dehydrogenase and peroxidase, showed similar expression patterns (Fig. 6), while one peroxidase was highly induced during all the time after infection in both the HR and HS grapes (Fig. 2c). Finally, four catalases were identified as being up-regulated only in the HS grape at 12 and 24 hpi (Fig. 6). Together, these data reveal that the expression of genes involved in phenylpropanoid and flavonoid pathways is extremely dynamic in grape after E. ampelina infection.
Phytohormone signaling related genes
Expression of multiple phytohormone signaling related genes were also affected by E. ampelina infection (Fig. 6). For example, an ETH signal transduction-related gene (ERF1) was up-regulated in both genotypes but with the delay in the HR grape. Eight TIFY proteins, three of which were jasmonate ZIM-domain (JAZ) genes, which function as repressors of JA signaling were induced in the HR grape at 24 hpi and in the HS grape at 72 hpi. In addition, jasmonate signal-related genes, lipoxygenases (LOX3), allene oxide cyclase (AOC4) and allene oxide synthase (AOS), showed similar expression patterns (Fig. 6). Disease susceptibility 1 (EDS1) and pathogen-related protein 1 (PR1) are genes associated with SA mediated defense responses, and were up-regulated in both grape varieties. In addition, three genes related to the ABA signaling pathway were induced earlier in the HS grape (Fig. 6). Our results also highlighted the involvement of GA in grape defense responses to E. ampelina as beta-glucosidase (BGL) expression varied substantially during E. ampelina infection (Fig. 6).