Enzyme activity analysis
To elucidate the early physiological response, the ‘Seli’ and ‘Cuiguan’ samples were collected after inoculation with C. fructicola at 0, 6, and 24 h and analyzed. The enzyme activity illustrated that the polyphenol oxidase (PPO) activity, and H2O2 content in ‘Seli’ leaves were notably higher than those in ‘Cuiguan’ leaves at 0, 6, and 24 h after inoculation (Fig. 2a and b). Furthermore, peroxidase (POD) activity was remarkably lower in ‘Seli’ leaves than in ‘Cuiguan’ leaves at 0, 6, and 24 h after inoculation (Fig. 2c).
Differentially expressed genes in ‘Seli’ and ‘Cuiguan’ in response to C. fructicola infection
To determine the molecular basis for ‘Seli’ resistance to anthracnose, samples were collected from leaves inoculated with C. fructicola (treatment) and sterile water (control) at 6 and 24 h. A total of 24 sample libraries were constructed, each with 40–55 million reads. The GC content and the number of repeat sequence reads were calculated using FastQC software (Table S1 in Additional File 1). More than 90% of the clean reads were mapped to the reference genome of P. pyrifolia (Gao et al., 2021), thus indicating that the quality of the transcriptome sequencing data was reliable. Principal component analysis, and Pearson’s correlation coefficient analysis results showed good reproducibility for each treatment (Fig. 3a and b). The total mapped reads of all genes were used for differential expression analysis using DESeq |log2(fold change)| ≥2 and false discovery rate FDR ≤ 0.05. A total of 3186 differentially expressed genes (DEGs) were identified in ‘Seli’ and ‘Cuiguan’ at 6 and 24 h after inoculation (Fig. 3c). Comparative analysis of DEGs between the two C. fructicola infection time points in ‘Seli’ revealed that they shared 145 DEGs (i.e. SL-6 and SL-24) (Fig. 3d). A similar comparison for ‘Cuiguan’ revealed 131 common genes at the two time points (Fig. 3d). In addition, the DEGs were analyzed between ‘Seli’ and ‘Cuiguan’ at 6 and 24 h post-infection, and we found that only 24 DEGs overlapped across all treatment samples (Fig. 3d).
Functional annotation of DEGs and pathway enrichment analysis
To fully understand the biological process of the identified DEGs in ‘Seli’ and ‘Cuiguan’ at 6 and 24 h after C. fructicola infection, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed. GO enrichment analysis of the DEGs revealed that there were six common pathways in the comparisons of CG-D versus CG-CF and SL-D versus SL-CF, which included response to hypoxia, response to ROS, reduction in ROS levels, oxylipin biosynthetic process, phenylpropanoid biosynthesis, and secondary metabolite biosynthetic process. In addition, the DEGs of SL-D versus SL-CF were enriched in response to chitin, response to wound, and immune effector processes (Fig. 4a and b). The KEGG enrichment results revealed that most DEGs of CG-D versus CG-CF and SL-D versus SL-CF were enriched in the biosynthesis of secondary metabolites and metabolic pathways. However, according to the enrichment pathway results, MAPK signaling, and flavonoid biosynthesis pathways were uniquely enriched in SL-D versus SL-CF (Fig. 4c and d). We speculated that the DEGs involved in the pathways of secondary metabolites, biosynthetic processes, and MAPK signaling pathway are closely related to ‘Seli’ resistance to C. fructicola infection.
Genes potentially involved in ‘Seli’ resistance to C. fructicola infection
We further analyzed the gene expression levels in four KEGG pathways: MAPK signaling pathway, phenylpropanoid biosynthesis, alpha-linolenic acid metabolism, and plant hormone signal transduction. The plant MAPK signaling pathway plays a pivotal role in plant disease resistance [15, 19]. Herein, we revealed that genes related to the PTI pathway were only induced in ‘Seli,’ which included leucine-rich repeat (LRR) receptor-like kinase (FLS2), BRI1-associated receptor kinase 1 (BAK1), MPK3, WRKY-type transcription factor 29 (WRKY29), oxidative signal inducible 1 (OXI1), MAP kinase substrate 1 (MKS1), and NADPH oxidase (RBOHD) (Fig. 5a). We inferred that the expression of these early defense genes in ‘Seli’ could induce ROS accumulation and cell death [11], thus restricting C. fructicola expansion in the early stage.
Phenylpropanoid biosynthesis is an important secondary metabolism pathway in plant disease resistance [20]. We found that the expression of genes related to flavonoid biosynthesis, including 4-coumarate:coenzyme A ligase (4CL), phenylalanine ammonia lyase, and p-coumarate 3-hydroxylase (C3’H), was remarkably upregulated in ‘Seli’ and ‘Cuiguan’ leaves after C. fructicola infection. However, the expression of genes related to fatty acid and flavonoid biosynthesis, including fatty acid desaturase, cinnamon alcohol dehydrogenase 5 (CAD5), anthocyanidin reductase (ANR), leucoanthocyanidin reductase (LAR), and two shikimate hydroxycinnamoyl transferases (HCT), was substantially upregulated in ‘Seli’ when inoculated with C. fructicola at 6 h (Fig. 5b).
Furthermore, transcription factors play an important role in the plant’s response to abiotic and biotic stresses by regulating the plant’s immune defense system [21]. In this study, we found that WRKY6, WRKY75, and WRKY76 were remarkably upregulated in ‘Seli’ and ‘Cuiguan’ at 6 and 24 h after C. fructicola infection, whereas WRKY9, WRKY50, and WRKY72 were particularly upregulated in ‘Seli’. Other transcription factors such as ERF95, ERF14, bHLH030, and MYB108 were also upregulated after C. fructicola infection (Fig. 5c).
Genes involved in different plant hormone signal transduction pathways were also characterized. We found that transcriptional repressors such as jasmonate zim-domain (JAZ) in JA signaling as well as SCF-type E3 ligase complex (GID2), and DELLA in the GA signaling pathway were upregulated after inoculation with C. fructicola, and their expression in ‘Cuiguan’ was remarkably greater than that in ‘Seli’. AOS is the second enzyme in the biosynthesis of the plant defensive hormone JA [22]. We found that the expression of AOS1 (EVM0031709) was upregulated in ‘Seli’ and ‘Cuiguan’ at 6 h, whereas the expression of AOS1 was specifically induced in the resistant variety of ‘Seli’ at 24 h after inoculation (Fig. 5d).
Weighted correlation network analysis (WGCNA) for the DEGs of ‘Seli’ and ‘Cuiguan’ in response to C. fructicola infection
To determine the gene regulatory network of ‘Seli’ and ‘Cuiguan’ in response to C. fructicola infection, a weighted gene coexpression network was constructed based on 3186 DEGs identified in the RNA-seq data. A network heatmap plot was constructed incorporating different gene dendrograms and modules to visualize the topological overlap matrix of the DEGs post-infection (Fig. 6a). In total, the network was divided into 19 modules according to the correlations between the modules and samples (Fig. 6b). The expression of genes in the brown module was upregulated in ‘Seli’ and downregulated in ‘Cuiguan’ (Fig. 6c). We then analyzed the biological processes of the DEGs in the brown module and found that these DEGs were enriched in the plant–pathogen interaction and MAPK signaling pathway (Fig. 6d). We further revealed that these DEGs were highly related to C. fructicola infection. Combining the KEGG enrichment results and gene function prediction, FLS2, MPK3, MKS1, and WRKY29 in the brown module appeared to be the hub genes that play important roles in plant–pathogen interaction (Fig. 6e).
Validation of RNA-seq data via quantitative reverse transcription polymerase chain reaction (qRT–PCR)
To confirm the quality of the transcriptome data, qRT–PCR was conducted for the nine candidate genes involved in phenylpropanoid metabolism, and the MAPK signaling pathway was also validated via qRT–PCR. The expression of these genes such as BAK1, FLS2, WRKY29, MPK3, and MKS1 were upregulated in ‘Seli’ at 6 and 24 h after C. fructicola inoculation, whereas their expression in ‘Cuiguan’ was only upregulated at 6 h after inoculation. Meanwhile, the expression of male discoverer 1-interacting receptor-like kinase 2 (MIK2) was upregulated in ‘Seli’ and ‘Cuiguan’ at 24 h after inoculation. Furthermore, the expression of calmodulin-like protein 19 (CML19), calcium-dependent protein kinase (CDPK), and CAD5 was induced and upregulated in ‘Seli’ leaves after C. fructicola inoculation. Thus, all selected genes showed similar expression patterns as those of the RNA-seq data (Fig. 2a, Fig. 6, Table S2 in Additional File 1).