Transcriptome and Metabolome Analyses Reveal Response Mechanisms to Alternaria brassicicola-Induced Black Spot Disease in Diverse Chinese Cabbage Genotypes

Abstract

Chinese cabbage (Brassica rapa L. ssp. pekinensis) is an important food crop. However, its growth and development are commonly impacted by black spot disease. To examine the response mechanisms of Chinese cabbage to black spot disease, transcriptome and metabolome sequencing were performed on the leaves of Chinese cabbage genotypes J405 (resistant) and B214 (susceptible), 48 h post-infection (hpi) with Alternaria brassicicola. Expression of essential genes in the jasmonic acid, cytokinin, and auxin signaling pathways of both Chinese cabbage genotypes was inhibited. The expression of the pathogenesis-related protein 1 (PR1) gene mediated by the salicylic acid pathway is inhibited in the Chinese cabbage genotype B214. The basic endochitase B (CHIB) gene in the ethylene pathway of both Chinese cabbage genotypes was upregulated. The accumulation of reactive oxygen species in the disease spots of Chinese cabbage genotype J405 was greater than in genotype B214. The respiratory burst oxidase (RBOH) gene in the reactive oxygen species metabolic pathway was significantly upregulated in genotype J405, while no change was observed in genotype B214. We found that oxidation-reduction-related genes such as type-2 peroxiredoxin genes, NADPH-dependent thioredoxin reductase genes, glutathione peroxidase genes, and glutathione S-transfer genes were differentially expressed across both Chinese cabbage genotypes at 48 hpi. Metabolomics demonstrated that delta-tocopherol and S-hexyl glutathione were all downregulated in genotype J405, while they were upregulated in genotype B214. This approach also identified differential expression of genes in the carotenoid biosynthesis pathway, the glycinebetaine biosynthesis pathway, as well as in the specific sulfur glycoside metabolism pathway. These findings indicate that ethylene signaling is important in the hormone signaling regulatory network-mediated disease resistance and defense in Chinese cabbage. When facing pathogen infection, hormone transduction pathways associated with growth and development in Chinese cabbage are inhibited. The accumulation of reactive oxygen species and the outbreak of various secondary metabolites may endow the Chinese cabbage genotype J405 with increased resistance to black spot disease.

1. Introduction

Chinese cabbage (Brassica rapa L. ssp. pekinensis) is a critical crop globally, noted for its rich nutrients, minerals, and dietary fiber, with a high yield [1,2]. However, it is often impacted by black spot disease during its production, caused by Alternaria species. Currently, five reported pathogens can infect Chinese cabbage to trigger black spot disease, namely Alternaria brassicae, Alternaria brassicicola, Alternaria raphani, Alternaria japonica, and Alternaria alternata. Among them, A. brassicae and A. brassicicola are the most widespread. They can harm the growth of the host, cause vegetable decay and spoilage during storage, and limit seed germination [3,4]. A. brassicicola is a necrotrophic pathogen that can use dead host tissues for growth and proliferation, often producing low molecular weight secondary metabolites with diverse structures, including cyclosporin, to kill host cells and obtain nutrients. Diseased plants exhibit necrotic spots on leaves and stems and gradually decay [5,6]. Under the long-term co-evolution with pathogens, plants have achieved two levels of disease resistance and defense mechanisms [7]. Plants recognize pathogen-associated molecular patterns (PAMPs) through molecular pattern recognition receptors (PRRs) on cell surfaces, which can initiate PAMP-triggered immunity (PTI) [8]. PTI activates disease resistance signal transduction networks made up of multiple signaling molecules, such as G protein, Ca²⁺, mitogen-activated protein kinase (MAPK), reactive oxygen species (ROS), jasmonic acid (JA), salicylic acid (SA), and ethylene (ET) pathways. Disease resistance and defense-associated genes are induced in plants, while secondary metabolites associated with disease resistance are produced to limit pathogen invasion [9]. However, some pathogens can inhibit PTI by secreting toxic effectors, allowing re-invasion of the host plant. Accordingly, plants can activate effector-triggered immunity (ETI) by recognizing the toxic effectors through resistance proteins [10]. ETI activates disease resistance signaling in plants, causing local cellular hypersensitive response (HR) at the infection site, resisting pathogen infection and further spread [11]. This local HR can induce defense gene expression across the entire plant, triggering systemic acquired resistance (SAR) [12]. To comprehend how Chinese cabbage responds to black spot disease, transcriptome and metabolome sequencing were conducted on the leaves of Chinese cabbage genotypes J405 and B214 at 48 h post-infection (hpi), and differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) were evaluated. This study offers valuable insights into the molecular foundation of Chinese cabbage responses to A. brassicicola infection and a comprehensive understanding of plant-pathogen interactions.

2. Materials and Methods

2.1. Materials and Experimental Treatment

Two-leaf Chinese cabbage plants were inoculated with 10 µL of spore suspension (1 × 10⁵/mL) of A. brassicicola (Institute of Plant Protection, Tianjin Academy of Agricultural Sciences, Tianjin, China) in the center of each leaf and covered with plastic wrap. The plants were placed in an artificial climate room (25 °C, relative humidity 96%) in the dark for 24 h, then moved to the light for 8 h, followed by darkness for 16 h.

The observations and statistics related to disease severity were performed on six Chinese cabbage inbred lines, J405, B214, HB5-D, HB6-D, HB7-D, and 904A, which were inoculated with A. brassicicola 1 day, 3 days, and 5 days after inoculation.

The leaves of Chinese cabbage inbred lines J405 and B214 were inoculated with A. brassicicola for 48 hpi as the experimental group, and 48 h after inoculation with sterile water operated as the control group. All treatment groups consisted of three biological replicates. The leaves of each treatment group were flash-frozen with liquid nitrogen and stored at −80 °C for subsequent transcriptome and metabolome analysis.

2.2. Methods for Identification of Plant Disease Resistance

Classification of disease severity was made according to the area of disease spots on leaves after inoculation with pathogens. Nine levels were identified, including level 0 (no symptoms), level 1 (lesion area accounts for <5% of the total leaf area), level 3 (lesion area accounts for between 6% and 10% of the total leaf area), level 5 (lesion area accounts for between 11% and 20% of the total leaf area), level 7 (lesion area accounts for between 21% and 50% of the total leaf area), and level 9 (lesion area accounts for >51% of the total leaf area). A disease index was employed to assess the disease resistance grade.

Disease index = ∑ (number of diseased leaves at all levels × disease level)/(total number of investigated leaves × the highest level) × 100.

Plants with disease severity scores of 0, 1, and 3 were categorized as resistant (0 was fully resistant, while 1 and 3 were partially resistant); scores of 5 were categorized as partially susceptible; and scores of between 7 and 9 were categorized as susceptible.

2.3. Trypan Blue Staining

Plant leaves were immersed in a 0.4% trypan blue dye solution, boiled in water for 3 min, and incubated overnight at room temperature. The plant tissue was soaked in 75% ethanol for 30 min, and this was repeated three times. Photographs were taken after soaking in 95% ethanol until completely decolorized.

2.4. Reactive Oxygen Species Staining

To detect the superoxide (O₂·⁻) radicals, plant leaves were immersed in 0.1% nitroblue tetrazolium (NBT) dye solution in the dark at room temperature for 3 h. The solution was decanted off, anhydrous ethanol was added for incubation in a water bath at 65 °C for 25 min, decolorized until transparent, and photos were taken [13].

2.5. RNA-Seq Experiments

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Samples were treated with TURBO DNase I (Ambion, Austin, TX, USA) for 30 min and purified using an RNeasy^® Plant MiniKit (QIAGEN, Hilden, Germany). RNA sequencing libraries were constructed with a TruSeq RNA sample Prep V2 kit (Illumina, San Diego, CA, USA) following the manufacturer’s directions. The quality and size of cDNA libraries were examined using the Agilent 2200 TapeStation system (Agilent, Santa Clara, CA, USA) prior to sequencing. cDNA libraries were sequenced using the Illumina NovaSeq 6000 under the 150-cycle paired-end sequencing protocol (Biomark, Beijing, China).

2.6. Transcriptomic Data Analysis

The reference genome (Brara_Chiifu_V3.5) and corresponding annotation files were obtained from the Brassicaceae Database (http://www.brassicadb.cn/#/Download/) (accessed on 7 November 2023) and used for genome mapping and annotation. Raw reads were filtered using Fastp and mapped to the reference genome with Hisat2 [14,15]. Fragments per kilobase of transcript per million mapped reads (FPKM) were calculated using Stringtie [16]. DEGs between treatment groups were characterized using the R package ‘Deseq2’ with thresholds of |log₂(fold change [FC])| ≥ 1 and false discovery rate (FDR) < 0.05 [17]. The R package ‘WGCNA’ was utilized for weighted gene co-expression network analysis [18]. AgriGO (http://systemsbiology.cau.edu.cn/agriGOv2/#) (accessed on 7 November 2023) was employed for Gene Ontology (GO) term enrichment analysis. Metabolic pathway analysis was performed using KEGG (https://www.kegg.jp/) (accessed on 7 November 2023) and Mapman [19].

2.7. Validation of Candidate Genes via Reverse Transcription Quantitative PCR (RT-qPCR)

Primers in the exon region of candidate genes were designed with Primer 5 and the NCBI database (Supplementary Table S1). Reverse transcription reactions were performed using 2 µg of total RNA and TaqMan Reverse Transcription Reagent (Applied Biosystems, Sparta, NJ, USA). Each 20 µL reaction encompassed 10 µL of Premix Ex Taq (RR420A, Takara, Kyoto, Japan), 0.5 µL of each forward and reverse primer (10 μmol/L), 7 µL of double distilled water, and 2 µL of cDNA. The thermocycling approach consisted of a denaturation step at 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 20 s. The relative expression level of each gene was calculated using the 2^−∆∆Ct method [20], with EF-1α serving as the internal reference gene. All RT-qPCR reactions were conducted in technical triplicate.

2.8. Metabolites Extraction and LC-MS/MS Analysis

The samples were freeze-dried in a vacuum freeze-dryer and pulverized in a mixing mill with zirconia beads at 45 Hz for 1 min. The lyophilized powder (50 mg) was dissolved in 1 mL mixture of methanol/acetonitrile/water (2:2:1, v/v/v) and vortexed for 30 s, and the samples were placed in a refrigerator overnight at 4 °C. After centrifugation at 12,000 rpm for 15 min, the extracts were filtered (SCAA-104, pore size 0.22 μm; ANPEL, Shanghai, China) to obtain the samples for LC-MS/MS analysis. In addition, an equivalent amount of 10 µL was aspirated from each sample’s extraction mixture and mixed homogeneously as QC samples.

The LC/MS system for metabolomics analysis comprised a Waters Acquity I-Class PLUS ultra-high performance liquid tandem Waters Xevo G2-XS QTOF high-resolution mass spectrometer (Waters, Milford, MA, USA). The column was a Waters Acquity UPLC HSS T3 column and used an injection volume of 1 μL. Parameters were as follows in the positive ion mode: mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: 0.1% formic acid acetonitrile. Parameters in the negative ion mode were as follows: mobile phase A: 0.1% formic acid aqueous solution; mobile phase B: 0.1% formic acid acetonitrile. A Waters Xevo G2-XS QTOF high-resolution mass spectrometer collected primary and secondary mass spectrometry data in MSe mode using MassLynx V4.2 (Waters, Milford, MA, USA). The low collision energy was 2 V, the high collision energy range was 10 to 40 V, and the scanning frequency was 0.2 s for a mass spectrum. The parameters of the ESI ion source were as follows: capillary voltage: 2000 V (positive ion mode) or 1500 V (negative ion mode); cone voltage: 30 V; ion source temperature: 150 °C; desolvent gas temperature: 500 °C; backflush gas flow rate: 50 L/h; desolventizing gas flow rate: 800 L/h.

2.9. Metabolomic Data Analysis

The raw data collected using MassLynx V4.2 was processed using Progenesis QI software V3.0 for peak extraction, peak alignment, and other data processing operations according to the Progenesis QI software online METLIN database and Biomark’s library for identification. Theoretical fragment identification and mass deviation were within 100 ppm. After normalizing the original peak area information with the total peak area, a follow-up analysis was performed. Principal component analysis and Spearman correlation analysis were employed to assess the repeatability of samples within the group and control samples. The identified compounds were queried for classification and pathway information in KEGG, the human metabolome database (https://hmdb.ca/, accessed them on 7 November 2023), and lipidmaps databases (https://www.lipidmaps.org) (accessed them on 7 November 2023). According to the grouping information, difference multiples were calculated and compared, and t-tests were used to determine the significance of differences according to the p-value of each compound. The R package ropls was used to perform OPLS-DA modeling, and 200 permutation tests were performed to verify the model’s reliability. The VIP value of the model was computed using multiple cross-validations. The method of integrating the difference multiple, p-value, and VIP value of the OPLS-DA model was utilized to screen differential metabolites. The DAMs screening criteria were FC > 1, p-value < 0.05, and VIP > 1. The significance of the different metabolites in the KEGG pathway enrichment was calculated using a hypergeometric distribution test [21,22].

3. Results

3.1. Black Spot Disease Severity across Diverse Chinese Cabbage Genotypes

Observations across six Chinese cabbage genotypes inoculated with A. brassicicola revealed that none had spots visible at 1 day post-inoculation (dpi). Chinese cabbage genotypes B214 and HB5-D reached level 3 disease severity, while the others reached level 1 disease severity at 3 dpi. The Chinese cabbage genotype J405 exhibited level 1 disease severity, HB6-D and HB7-D showed level 3 disease severity, and the remaining genotypes were level 5 disease severity at 5 dpi. Microscopic observation of the plant indicated that pathogen spores formed numerous mycelia on the leaves and could infect the stem. The affected leaves had water-stained dark brown spots, which expanded, encompassing a yellow halo and gray mold on the spots (Figure 1).

3.2. DEGs in Chinese Cabbage Genotypes J405 and B214 in Response to Black Spot Disease

Transcriptome sequencing was conducted on Chinese cabbage leaves from genotypes J405 and B214 at 48 hpi. A total of 12 sequencing samples were extracted, with an average read count of 46.90 MB and an average sequencing data size of 6.89 GB per sample. The average Q30 value for each sample was 96.63% (Supplementary Table S2). Compared to Chinese cabbage inoculated with sterile water, genotype J405 possessed 5003 DEGs and B214 had 5727 DEGs (Figure 2A). Ten DEGs were selected randomly for RT-qPCR validation, with an R² of 0.9 (Supplementary Figure S1). This indicated that the sequencing quality and analysis of DEGs were consistent and could be further investigated. Analysis of these DEG sets uncovered a crossover in the differential gene sets across genotypes. The number of DEGs was 1456 in Chinese cabbage genotype J405, and 2180 in genotype B214. The two shared genotypes had 3547 common DEGs, accounting for 49.4%. The number of upregulated and downregulated DEGs in genotype B214 was higher than in J405 (Figure 2B), indicating that Chinese cabbage genotype B214 had a more robust response to pathogen infection than J405 at the transcriptional level.

Based on GO and KEGG annotation, the DEGs of Chinese cabbage genotype J405 are primarily localized in the lipid biosynthetic process (GO: 0008610), pectinesterase activity (GO: 0030599), cell wall modification (GO: 0042545), phosphorelay signal transduction system (GO: 0000160), response to hormone (GO: 0009725), autophagy (GO: 0006914), and glutathione metabolic process (GO: 0006749) genes. These genes are predominantly involved in pathways such as transcription factors (KEGG: 03000), plant hormone signal transduction (KEGG: 04075), replication and repair (KEGG: 09124), biosynthesis of secondary metabolites (KEGG: 09110), fatty acid elongation (KEGG: 00062), cytochrome P450 (KEGG: 00199), and cutin, suberine, and wax biosynthesis (KEGG: 00073). The DEGs set found in Chinese cabbage genotype B214 includes those associated with lipid biosynthetic process (GO: 0008610), aminoacyl-tRNA ligase activity (GO: 0004812), protein peptidyl-prolyl isomerization (GO: 0000413), pectinesterase activity (GO: 0030599), cell wall modification (GO: 0042545), and phosphorelay signal transduction system (GO: 0000160) genes. These genes are primarily involved in pathways such as cytochrome P450 (KEGG: 00199), glucosinolate biosynthesis (KEGG: 00966), DNA replication proteins (KEGG: 03032), plant hormone signal transduction (KEGG: 04075), and fatty acid biosynthesis (KEGG: 00061) (Figure 2). A total of 33 kinase genes in the MAPK pathway and 431 TFs responded to black spot disease at the transcriptional level (Supplementary Table S3 and Figure S2).

3.3. Response of Genes Related to Hormone Signaling Pathways in Chinese Cabbage to Infection by A. brassicicola

Chinese cabbage genotypes J405 and B214 activate JA, SA, ET, brassinolide (BR), abscisic acid (ABA), and other hormone signal transduction pathways at 48 hpi. A total of six key genes were identified in the ET signaling pathway, including the serine/threonine protein kinase (CTR1) gene being downregulated in genotypes J405 and B214. In contrast, the defense gene CHIB was upregulated in both genotypes. Ethylene receptor (ETR), ethylene-insensitive protein 3 (EIN3), and ethylene-responsive transcription factor 1 (ERF1) genes were specifically expressed in Chinese cabbage genotype B214, with no significant change observed in genotype J405. The brassinosteroid-insensitive 1-associated receptor kinase 1 (BAK1) gene in the BR pathway was upregulated in Chinese cabbage genotype B214 (log₂FC: 5.87), while no significant alterations were observed in genotype J405. Other genes in the BR pathway exhibited similar expression patterns across both Chinese cabbage genotypes. The key genes in the ABA pathway of genotypes J405 and B214 exhibited similar expression patterns. The three genes, ABA receptor PYR/PYL family (PYL), protein phase 2C (PP2C), and serine/threonine protein kinase (SNRK2), were downregulated in both Chinese cabbage genotypes. However, the ABA-responsive element binding factor (ABF) gene was only downregulated in genotype B214 (log₂FC: −1.04). The gene encoding coronate-intensive protein 1 (COI-1) in the JA signaling pathway was downregulated in Chinese cabbage genotypes J405 and B214, while the gene encoding jasmonate ZIM domain-containing protein (JAZ) was upregulated in both genotypes. These results indicated that the JA signaling pathway of Chinese cabbage genotypes with different black spot resistance was inhibited in response to pathogen infection. The regulatory protein (NPR1) gene in the SA signaling pathway was upregulated, while the transcription factor (TGA) gene was downregulated in genotypes J405 and B214 at 48 hpi. The gene PR1 was downregulated only in Chinese cabbage genotype B214 (log₂FC: −3.42). The expression of disease resistance protein genes facilitated by the SA pathway was inhibited in Chinese cabbage genotype B214, while it remained consistent in genotype J405.

The expression patterns of critical genes in the cytokinin and auxin signaling pathways were similar across both Chinese cabbage genotypes at 48 hpi. However, the change of most DEGs in genotype B214 was greater than in genotype J405. The Arabidopsis histidine kinase 2/3/4 (AHK) gene and the histidine-containing phototransfer protein (AHP) gene were upregulated in both Chinese cabbage genotypes. The two-component response regulator ARR-B family (ARR-B) gene and two-component response regulator ARR-A family (ARR-A) gene in the nucleus were downregulated in Chinese cabbage genotypes J405 and B214. The auxin flux carrier (AUX1) and transport inhibitor response 1 (TIR1) genes in the auxin signaling pathway were upregulated in both Chinese cabbage genotypes. The auxin-response protein (IAA) and auxin-response factor (ARF) genes were downregulated in Chinese cabbage genotypes J405 and B214. The downstream genes in this pathway, auxin-responsive GH3 family protein (GH3) and SAUR family protein (SAUR), were downregulated in both Chinese cabbage genotypes (

Table 1. Expression of genes associated with hormone signaling pathways in Chinese cabbage genotypes J405 and B214 at 48 hpi.

KOID	GeneID	Annotation	J405-48h-DEGs	B214-48h-DEGs
K13463	BraA05g006060.3.5C	COI-1, coronatine-insensitive protein 1, JA	−1.33	−2.37
K13464	BraA07g030900.3.5C	JAZ, jasmonate ZIM domain-containing protein, JA	2.90	4.30
K14431	BraA10g029510.3.5C	TGA, transcription factor TGA, SA	−2.41	−2.26
K14508	BraA01g016900.3.5C	NPR1, regulatory protein NPR1, SA	2.06	3.07
K13449	BraA03g012320.3.5C	PR1, pathogenesis-related protein 1, SA	-	−3.42
K14509	BraA08g035470.3.5C	ETR, ethylene receptor, ET	-	−1.28
K14514	BraA07g029620.3.5C	EIN3, ethylene-insensitive protein 3, ET	-	1.71
K14516	BraA03g016550.3.5C	ERF1, ethylene-responsive transcription factor 1, ET	-	−2.78
K14510	BraA10g032440.3.5C	CTR1, serine/threonine-protein kinase CTR1, ET	−2.53	−2.10
K14512	BraA05g003850.3.5C	MPK6, mitogen-activated protein kinase 6, ET	-	−3.62
K20547	BraA03g035780.3.5C	CHIB, basic endochitinase B, ET	1.84	2.42
K13946	BraA02g025050.3.5C	AUX1, auxin influx carrier, Auxin	3.96	4.46
K14484	BraA10g002540.3.5C	IAA, auxin-responsive protein IAA, Auxin	−2.59	−3.16
K14486	BraA04g025570.3.5C	ARF, auxin response factor, Auxin	−3.11	−5.07
K14487	BraA03g020320.3.5C	GH3, auxin responsive GH3 gene family, Auxin	−2.44	−4.19
K14488	BraA01g003530.3.5C	SAUR, SAUR family protein, Auxin	−3.36	−4.53
K14485	BraA04g000640.3.5C	TIR1, transport inhibitor response 1, Auxin	1.90	2.10
K14490	BraA07g026540.3.5C	AHP, histidine-containing phosphotransfer protein, cytokinin	1.61	1.50
K14491	BraA03g038160.3.5C	ARR-B, two-component response regulator ARR-B family, cytokinin	−2.33	−2.92
K14492	BraA06g007350.3.5C	ARR-A, two-component response regulator ARR-A family, cytokinin	−4.00	−4.31
K14489	BraA03g042410.3.5C	AHK2_3_4, Arabidopsis histidine kinase 2/3/4, cytokinin	1.47	2.28
K14494	BraA02g017510.3.5C	DELLA, DELLA protein, Gibberellin	−4.83	−2.91
K16189	BraA01g008670.3.5C	PIF4, phytochrome-interacting factor 4 TF, Gibberellin	−1.73	−3.66
K12126	BraA03g024450.3.5C	PIF3, phytochrome-interacting factor 3 TF, Gibberellin	1.17	2.67
K14432	BraA07g023280.3.5C	ABF, ABA responsive element binding factor, ABA	-	−1.04
K14496	BraA08g013180.3.5C	PYL, abscisic acid receptor PYR/PYL family, ABA	−4.55	−5.10
K14497	BraA10g006150.3.5C	PP2C, protein phosphatase 2C, ABA	4.53	6.48
K14498	BraA03g003810.3.5C	SNRK2, serine/threonine-protein kinase SRK2, ABA	−1.88	−1.54
K14503	BraA01g002020.3.5C	BZR1_2, brassinosteroid resistant 1/2, BR	−2.58	−1.18
K14500	BraA03g044040.3.5C	BSK, BR-signaling kinase, BR	2.69	3.09
K14505	BraA01g004150.3.5C	CYCD3, cyclin D3, plant, BR	2.96	3.47
K13415	BraA01g000440.3.5C	BRI1, protein brassinosteroid insensitive 1, BR	−2.36	−1.31
K13416	BraA03g058830.3.5C	BAK1, brassinosteroid insensitive 1-associated receptor kinase 1, BR	-	5.87

). The expression of positive regulatory genes in the auxin pathway was downregulated. These findings indicated that the cytokinin and auxin signaling pathways in different disease-resistant Chinese cabbage genotypes were inhibited in response to pathogen infection.

3.4. Response of Genes Associated with Disease Resistance Pathways in Chinese Cabbage to Infection by A. brassicicola

We identified genes related to the disease resistance pathway and found that cyclic nucleotide-gated channel protein (CNGC) gene expression was significantly upregulated in Chinese cabbage genotype J405 (log₂FC: 5.30) but exhibited no change in genotype B214. Calcium receptor-related genes CALM, CML, and CPK were upregulated in both Chinese cabbage genotypes. The expression of RBOH, a crucial gene in reactive oxygen metabolism, was significantly upregulated in genotype J405 (log₂FC: 1.92) but was unchanged in genotype B214. The expression of the gene senescence-induced receptor-like serine/threonine-protein kinase (FRK1) was upregulated; however, PR1 was downregulated in both Chinese cabbage genotypes.

The genes encoding pto-interacting protein 1 (PTI1), suppressor of G2 allele of SKP1 (SUGT1), pathogen-induced protein kinase (PIK1), and 3-ketoacyl-CoA synthase (KCS) were downregulated in both Chinese cabbage genotypes. Disease resistance protein genes RPM1, RPS2, and HSP90B were upregulated in both Chinese cabbage genotypes. In addition to the shared genes, RAR1 (log₂FC: 1.18), RPS5 (log₂FC: 1.82), and CTSF (log₂FC: 1.54) are specifically upregulated in the J405 genotype (

Table 2. Expression of genes associated with disease resistance and defense pathways in Chinese cabbage genotypes J405 and B214 at 48 hpi.

KOID	GeneID	Annotation	J405-48h-DEGs	B214-48h-DEGs
K05391	BraA01g028830.3.5C	CNGC, cyclic nucleotide gated channel	5.30	-
K02183	BraA07g020630.3.5C	CALM, calmodulin	3.98	3.16
K13448	BraA07g024690.3.5C	CML, calcium-binding protein	3.93	3.32
K13412	BraA02g044280.3.5C	CPK, calcium-dependent protein kinase	2.53	3.76
K13447	BraA02g040970.3.5C	RBOH, respiratory burst oxidase	1.92	-
K16224	BraA08g034860.3.5C	FRK1, senescence-induced receptor-like serine/threonine-protein kinase	1.13	1.54
K13449	BraA06g003510.3.5C	PR1, pathogenesis-related protein 1	−3.57	−3.85
K20547	BraA03g035780.3.5C	CHIB, basic endochitinase B	1.84	2.42
K13436	BraA05g013950.3.5C	PTI1, pto-interacting protein 1	−3.95	−3.74
K13457	BraA10g002960.3.5C	RPM1, disease resistance protein	2.92	2.24
K13458	BraA02g016130.3.5C	RAR1, disease resistance protein	1.18	-
K12795	BraA01g014280.3.5C	SUGT1, suppressor of the G2 allele of SKP1	−3.08	−3.20
K09487	BraA08g020900.3.5C	HSP90B, heat shock protein 90 kDa beta	1.21	1.38
K13459	BraA08g031690.3.5C	RPS2, disease resistance protein	1.53	1.21
K13430	BraA09g003180.3.5C	PBS1, serine/threonine-protein kinase	-	−1.09
K13460	BraA06g009450.3.5C	RPS5, disease resistance protein	1.82	-
K18873	BraA06g005150.3.5C	PIK1, pathogen-induced protein kinase	−4.44	−2.74
K16226	BraA03g028120.3.5C	RPS4, disease resistance protein	−1.43	−1.24
K01373	BraA03g047330.3.5C	CTSF, cathepsin F	1.54	-
K15397	BraA03g057320.3.5C	KCS, 3-ketoacyl-CoA synthase	−2.09	−2.03
K13423	BraA02g002290.3.5C	WRKY25, WRKY transcription factor 25	3.08	2.72
K02358	BraA03g050050.3.5C	Tuf, elongation factor Tu	2.21	2.71
K04368	BraA03g012900.3.5C	MAP2K1, mitogen-activated protein kinase 1	2.28	2.03
K13414	BraA03g026820.3.5C	MEKK1, mitogen-activated protein kinase 1	−2.58	−2.52
K14512	BraA03g023140.3.5C	MPK6, mitogen-activated protein kinase 6	−1.51	−2.47
K18835	BraA03g013060.3.5C	WRKY2, WRKY transcription factor 2	1.18	-
K13416	BraA03g058830.3.5C	BAK1, brassinosteroid insensitive 1-associated receptor kinase 1	-	5.87
K18834	BraA03g042400.3.5C	WRKY1, WRKY transcription factor 1	-	1.04

). We detected genes with NBS-LRR conserved domains for disease resistance and defense, finding 36 differentially expressed disease resistance genes with NBS-LRR domains, of which 20 were differentially expressed in both Chinese cabbage genotypes. The J405 genotype specifically expressed six genes containing this domain, namely TIR-NBS-LRR (BraA08g021450.3.5C), CC-NBS-LRR (BraA06g009450.35C, BraA06g003460.3.5C, BraA01g004810.3.5C), CC-NBS (BraA01g037730.3.5C), and NBS-LRR (BraA03g004710.3.5C) (Supplementary Table S4).

3.5. DAMs in Chinese Cabbage Genotypes J405 and B214 in Response to Black Spot Disease

Untargeted metabolomics was conducted on Chinese cabbage genotypes J405 and B214 leaves at 48 hpi. A total of 12 samples were extracted, and four groups of QC samples were used. A total of 9453 peaks were identified, corresponding to 2621 metabolites. A total of 6246 peaks were detected in the positive ion mode, corresponding to 1436 metabolites annotated. In negative ion mode, 3207 peaks were detected, and 1185 metabolites were annotated. OPLS-DA analysis demonstrated that the samples of each treatment group clustered well (Supplementary Figure S3).

Compared to Chinese cabbage inoculated with sterile water, there were 38 DAMs in the Chinese cabbage genotype J405 at 48 hpi, including S-hexyl glutathione, N-formyl-L-tyrosine, N-succinyl-2-amino-6-ketoximate, delta-tocopherol, dethiobiotin, and pantetheine. Among these, 21 DAMs were upregulated, and 17 DAMs were downregulated. These DAMs primarily involve neomycin, kanamycin, and gentamicin biosynthesis (KO00524), polyketide sugar unit biosynthesis (KO00523), pantothenate and CoA biosynthesis (KO00770), carbapenem biosynthesis (KO00332), biotin metabolism (KO00780), monoterpenoid biosynthesis (KO00902), isoquinoline alkaloid biosynthesis (KO00950), tyrosine metabolism (KO00350), and ubiquinone and terpenoid-quinone biosynthesis (KO00130), among others. There were 115 DAMs in the Chinese cabbage genotype B214 at 48 hpi, including N2-succinyl-L-ornithine, cyclopentolate, D-dethiobiotin, 5-hydroxyindoleacetic acid, and (S)-hydroxydecanoyl-CoA. Among these, 48 DAMs were upregulated, and 67 DAMs were downregulated. These DAMs were primarily involved in isoquinoline alkaloid biosynthesis (KO00950), diterpenoid biosynthesis (KO00904), phenylalanine metabolism (KO00360), tropane, piperidine, and pyridine alkaloid biosynthesis (KO00960), fatty acid degradation (KO00071), nicotinate and nicotinamide metabolism (KO00760), and fructose and mannose metabolism (KO00051), among others (Figure 3).

3.6. Redox Substances Are Essential in Disease Resistance and Defense of Chinese Cabbage

The accumulation of reactive oxygen species in disease spots of genotype J405 is greater than in genotype B214 at 48 hpi (Figure 4A). The type-2 peroxiredoxin gene, NADPH-dependent thioredoxin reductase family gene, glutathione peroxide gene, glutathione S-transfer family gene, and 14 other genes were differentially expressed at 48 hpi (Figure 4B). The upregulated expression of the glutathione peroxidase gene (BraA03g022830.3.5C) was identified in both Chinese cabbage genotypes (J405: log₂FC = 1.83, B214: log₂FC = 1.64). Six glutathione S-transferase family genes were detected, among which BraA02g020090.3.5C, BraA06g022560.3.5C, and BraA03g016350.35C were upregulated, while BraA07g027610.3.5C, BraA06g031710.3.5C, and BraA10g001520.3.5C were downregulated across both genotypes. The glutathione S-transferase gene (BraA04g022340.3.5C) was specifically downregulated in the J405 genotype (log₂FC: −1.80). Moreover, delta-tocopherol (pos_4213) was downregulated in the J405 genotype (log₂FC: −1.04), while it was upregulated in genotype B214 (log₂FC: 0.65). Hexylglutathione (pos_5316) was downregulated in the J405 genotype (log₂FC: −0.61) but upregulated in B214 (log₂FC: 1.61) (Figure 4C). The crucial genes and metabolites in the glutathione and tocopherol pathways were detected simultaneously in the metabolome and transcriptome, suggesting that redox substances such as glutathione and tocopherol might play important roles in disease resistance and defense.

3.7. Various Secondary Metabolites Play Important Roles in Disease Resistance and Defense of Chinese Cabbage

Key genes in various secondary metabolic pathways, including isoprene diphosphate biosynthesis, cycloartenol biosynthesis, carotenoid biosynthesis, p-coumaroyl CoA biosynthesis, and lignan biosynthesis, were differentially expressed relative to the control group. For example, two genes in the carotenoid biosynthesis pathway (BraA02g007030.3.5C: phytoene synthesis, BraA01g010600.3.5C: carotenoid cleavage dioxygenase) and three diterpene synthesis genes in the terpene biosynthesis pathway were differentially upregulated in both genotypes. The phenylalanine ammonia-lyase gene (BraA05g008230.3.5C) in the p-coumaroyl-CoA biosynthesis pathway and the isoprenyl diphosphate synthesis gene (BraA03g039690.3.5C) in the isoprenyl diphosphate biosynthesis pathway were differentially downregulated across both genotypes. In the glucosinolate metabolism pathway of Chinese cabbage, six genes, including the chorine monooxygenase gene in the glycinebetaine biosynthesis pathway, and four genes, including the myrosinase gene in the glucosinolate degradation pathway, were expressed at 48 hpi, with similar expression patterns in both genotypes. In addition to the genes common to Chinese cabbage genotypes J405 and B214, some DEGs specific to Chinese cabbage genotype J405 were identified, including three diterpene synthase genes in the terpene biosynthesis pathway (BraA03g036980.3.5C, BraA03g037000.3.5C, BraA10g021690.3.5C), three genes in the flavonoid biosynthesis pathway (BraA08g035240.3.5C: 3-ketoacyl CoA thiolase, BraA07g040000.3.5C: isoflavone reductase, BraA02g007020.3.5C: anthocyanin glutathione S-transferase), and seven genes in the glucosinolate biosynthesis pathway (Supplementary Table S5).

Secondary metabolite identification in different treatment groups at the metabolic level revealed the accumulation of multiple differential secondary metabolites. For instance, the content of dihydroshikonofuran (C18135, log₂FC: −1.08) in the ubiquinone and terpenoid-quinone biosynthesis pathways, as well as 2,6-dihydroxypseudooxynicotine (C15986, log₂FC: −2.13) in nicotinate and nicotinamide metabolism, were significantly downregulated in genotype B214, while there was no significant change in genotype J405. The levels of loganate (C01512, log₂FC: 1.91) in the monoterpenoid biosynthesis pathway and pantetheine (C00831, log₂FC: 1.47) in the pantothenate and CoA biosynthesis pathways were significantly upregulated in genotype J405, while there was no significant difference in genotype B214 (Supplementary Figure S4).

3.8. Association Analysis of Key Genes and Metabolites

WGCNA calculations were performed on DEGs across all samples, resulting in 17 modules (Figure 5A). The yellow module was significantly positively associated with the Chinese cabbage genotype J405 inoculation pathogen treatment group (Figure 5B). KEGG analysis of this module indicated that these genes were mainly involved in transcriptional regulation, protein binding, signal transduction, and other pathways (Figure 5C). It was determined that the DAMs were correlated with most genes in the yellow module, associating the genes in the yellow module with the DAMs in genotype J405 (Figure 5D). Numerous genes and metabolites in the glutathione metabolic pathway in both genotypes, a possible correlation regulatory network of the key metabolite S-hexyl glutathione (pos_5461) was plotted, with a Pearson correlation coefficient greater than 0.9 and significance less than 0.05 as the threshold (Figure 5E). A total of 45 genes were identified in the regulatory network, primarily phenylalanine ammonia-lyase, calcineurin-like phosphoesterase, diacylglycerol kinase family protein, sugar transporter family protein, glycoside hydrolase family protein, carboxylic ester hydrolase, glycosyltransferase family protein, and 3-ketoacyl-CoA synthase.

4. Discussion

Plants have evolved multifaceted defense systems to overcome various pathogen infections, primarily composed of molecular networks of multiple signal transduction pathways. To examine the disease resistance and defense mechanism of Chinese cabbage against black spot disease, we conducted transcriptome and metabolome sequencing on leaves of Chinese cabbage genotypes J405 and B214 at 48 hpi.

Plant hormones and their signal transduction networks are essential in disease resistance and defense systems. Among them, SA, JA, and ET are crucial for interactions between plants and pathogens. JA can enhance resistance to necrotrophic pathogens, whereas sensitivity to biotrophic pathogens increases [23,24]. ET is often synergistically involved with JA in inducing plant resistance to necrotrophic pathogens while antagonizing SA-mediated resistance to biotrophic pathogens. The JA and ET signaling pathways depend on ERF transcription factors to synergize in resisting necrotrophic pathogens [25,26,27]. This study found that both Chinese cabbage genotypes J405 and B214 activate different hormone signaling pathways such as SA, JA, and ET at 48 hpi. However, various Chinese cabbage genotypes had diverse hormone response approaches to overcome pathogen infection. The expression of PR1 mediated by the SA pathway is inhibited in genotype B214, which was not altered in J405. The JA signaling pathway was inhibited in both genotypes, but the disease resistance defense gene CHIB was upregulated. CHIB protein exerts antibacterial activity and can hydrolyze chitin and destroy fungal structures [28]. Therefore, Chinese cabbage coordinates JA and SA pathways to exert ET pathway-mediated disease resistance and defense. Moreover, hormones such as BR, ABA, auxin, and cytokinin participate in plant growth and development, and play important roles in disease resistance and defense [29]. There are many shared components between BR and PTI signaling pathways, including BAK1, BSK1, and BIK1. Moreover, FLS2 in the PTI pathway forms a heterodimer with BAK1 after sensing flg22 and activates downstream signaling pathways. This suggests that there may be crosstalk between BR and PTI signaling pathways [30]. In this study, the gene BAK1 of the BR pathway was specifically upregulated in Chinese cabbage genotype B214 (log₂FC: 5.87), while the gene was not affected in Chinese cabbage genotype J405. Other genes in the BR pathway exhibit the same expression pattern in both Chinese cabbage genotypes. These genes may serve as an important crossover point to finely regulate BR-mediated growth, development, and defense responses. Cytokinin and auxin are heavily involved in cell division, inducing organ differentiation and establishment. However, some studies have demonstrated that cytokinin and auxin are indirectly involved in the process of plant resistance to pathogen infection [31,32]. In this study, both Chinese cabbage genotypes inhibit cytokinin and auxin signaling pathways and limit growth and development, focusing on disease resistance and defense after infection. This suggests that various plant hormone signaling pathways are related to synergistically regulating the growth and development of Chinese cabbage, as well as balancing disease resistance and defense.

In addition to hormones, ROS are critical communicators. An appropriate concentration of ROS can inhibit the germination of pathogenic spores and limit the survival of pathogens. It can also induce plant resistance to diseases, promote cell wall lignification, and induce HR and SAR [33,34,35]. ROS can operate as second messengers to amplify stress signals in conjunction with Ca²⁺ activation of the MAPK pathway and activated MAPK can be transported to the nucleus for phosphorylation and activation of TFs to modulate resistance and defense gene expression [36,37]. The levels of ROS staining and the expression of ROS-related enzyme genes in genotype J405 were higher than those in B214. The Ca²⁺ receptor-related genes CALM, CML, and CPK are upregulated in both Chinese cabbage genotypes. The genes CNGC (log₂FC: 5.30) and RBOH (log₂FC: 1.92) were significantly upregulated in the Chinese cabbage genotype J405, while there was no alteration in genotype B214. Multiple kinase and TF family genes were identified to respond to black spot disease at the transcriptional level. Under typical environmental conditions, the production and clearance of ROS in plants are always in dynamic equilibrium. However, stress induces ROS accumulation in plants, leading to lipid peroxidation of the cell membrane and damage to the membrane system [38,39]. Protective enzymes and antioxidants accomplish the clearance of ROS in plants. The protective enzyme systems include superoxide dismutase, catalase, peroxidase, ascorbic acid peroxidase, and glutathione peroxidase. Antioxidants primarily include vitamin E, vitamin C, mannitol, and glutathione [40,41]. In this study, 14 redox-related genes, including the type-2 peroxiredoxin gene, NADPH-dependent thioredoxin reductase family genes, glutathione peroxidase gene, and glutathione S-transfer family genes, were differentially expressed across both genotypes of Chinese cabbage at 48 hpi. In the metabolome findings, we identified delta-tocopherol, S-hexyl glutathione, and hexylglutathione as downregulated in Chinese cabbage genotype J405 but upregulated in genotype B214. Therefore, Chinese cabbage genotype J405 may downregulate ROS scavenging enzyme levels and antioxidant metabolites while upregulating the expression of RBOH, increasing the ROS content in vivo, and enhancing resistance to black spot disease.

Secondary metabolites can be employed as mechanical and biochemical barriers to defend against pathogen infection and can be employed as signals to participate in plant disease resistance response [42]. We identified that glucosamine-1p, p-hydroxyphenylacetylglycine, canavanine, 5-hydroxyindoleacetic acid, methylergonovine, and dihydrozeatin-O-glucoside are differentially accumulated in both Chinese cabbage genotypes at 48 hpi. The diterpene synthase, phosphomevalonate kinase, isoflavone reductase, phenylalanine ammonia-lyase genes, and other key genes in the metabolite synthesis pathway were differentially expressed. Studies have shown that phytopreservin, saponin, nicotine, glucosinolide, isoflavonoids, alkaloids, and other compounds have antibacterial effects when interacting with pathogens and are involved in physiological processes such as HR and stress responses [43,44]. Therefore, the differential metabolites identified in this study and key enzyme genes in related synthesis pathways may be an important part of the chemical strategy for Chinese cabbage defense against black spot disease.

5. Conclusions

This study performed transcriptome and metabolome sequencing on Chinese cabbage genotypes J405 and B214 leaves at 48 hpi. The expression of key genes in the JA, cytokinin, and auxin signaling pathways of Chinese cabbage was found to be inhibited in response to infection with A. brassicicola. The ET signaling-mediated disease resistance defense pathway is activated. The genotype J405 can downregulate the antioxidant content of delta-tocopherol and S-hexyl glutathione while upregulating the expression of RBOH, enhancing the accumulation of reactive oxygen species at disease spots, and inhibiting pathogen infection. The potential correlation regulatory network of S-hexyl glutathione was obtained via association analysis. These findings offer a theoretical reference for understanding the resistance and defense mechanisms of Chinese cabbage against black spot disease and provide target genes for subsequent molecular breeding of Chinese cabbage for disease resistance.