Glucosinolate Profile and Glucosinolate Biosynthesis and Breakdown Gene Expression Manifested by Black Rot Disease Infection in Cabbage

Cabbage (Brassica oleracea var. capitata) is an economically important crop in the family Brassicaceae. Black rot disease is a top ranked cabbage disease, which is caused by Xanthomonas campestris pv. campestris (Xcc) and may reduce 50% crop loss. Therefore, we need a clear understanding of black rot disease resistance for sustainable disease management. The secondary metabolites, like Glucosinolate (GSL) presents in Brassica species, which plays a potential role in the defense mechanism against pathogens. However, there is little known about GSL-regulated resistance mechanisms and GSL biosynthesis and the breakdown related gene expression after black rot disease infection in cabbage. In this study, relative expression of 43 biosynthetic and breakdown related GSLs were estimated in the black rot resistant and susceptible cabbage lines after Xcc inoculation. Ten different types of GSL from both aliphatic and indolic groups were identified in the contrasting cabbage lines by HPLC analysis, which included six aliphatic and four indolic compounds. In the resistant line, nine genes (MYB122-Bol026204, MYB34-Bol017062, AOP2-Bo9g006240, ST5c-Bol030757, CYP81F1-Bol017376, CYP81F2-Bol012237, CYP81F4-Bol032712, CYP81F4-Bol032714 and PEN2-Bol030092) showed consistent expression patterns. Pearson’s correlation coefficient showed positive and significant association between aliphatic GSL compounds and expression values of ST5c-Bol030757 and AOP2-Bo9g006240 genes as well as between indolic GSL compounds and the expression of MYB34-Bol017062, MYB122-Bol026204, CYP81F2-Bol012237, CYP81F4-Bol032712 and CYP81F4-Bol032714 genes. This study helps in understanding the role of GSL biosynthesis and breakdown related genes for resistance against black rot pathogen in cabbage, which could be further confirmed through functional characterization either by overexpression or knock-out mutation.


Introduction
The bacterial species Xanthomonas campestris infects a wide range of Brassica species, including cabbage. Korea is among the larger cabbage producers and stands on fifth position in the world, worth approximately 66 million US$ from 2.12 million ton per year [1]. Black rot is a major disease in cabbage and was first identified in Korea in the 1970s [2]. It is a seed-borne disease and is caused by necrotrophic plant bacteria Xanthomonas campestris pv. campestris (Xcc) [3]. This pathogen is distributed frequently around the world and causes 50% economic loss [4]. The Xcc pathogen invades the xylem tissues after penetrating through hydathodes and wounded tissues, afterwards disease symptoms are developed in the host plants at warm and humid conditions [5]. Eleven races of the Xcc pathogen have been reported, which are proposed based on interaction between Xcc strains and differential Brassica cultivars following the gene-for-gene model [6,7]. Among them, races 1 and 4 were reported as the most virulent in cabbage [8]. Black rot disease management was attempted by using pathogen free seeds in cultivation, elimination of infected plant debris and cruciferous weeds, crop rotation and finally applying agrochemicals [9]. However, the use of resistant varieties at the farmers level will be the most sustainable way to control the disease [10].
Different defense reactions of host-pathogen such as resistance, susceptibility or lack of interaction in host plants are induced by the pathogen [11]. Glucosinolates (GSLs) were reported to show functions in plant defense mechanisms against pathogens [12,13]. GSLs are sulfur and nitrogen containing secondary metabolites broadly found in different Brassica species. They could be conferred as resistant against pathogens and insects [14]. Two important types of GSLs (aliphatic and indolic) are predominant in the crops of Brassicaceae and Arabidopsis [15,16]. GSLs are hydrolyzed by an endogenous myrosinase enzyme (β-thioglucoside glucohydrolase; EC 3.2.1.147) in ruptured cells and most of the hydrolyzed products seem to act as key regulators in tolerance reactions against Xcc pathogens [11,17]. The GSL hydrolyzed products, especially isothiocyanates (ITCs) are reported to have beneficial effects, like anti-carcinogenic and anti-oxidative activities for reducing risk of degenerative diseases in human health [18,19].
GSL content is varied within Brassica species or between cultivars due to allelic variation of GSL biosynthesis genes [20]. It has been noted that three loci (GSL-PRO, GSL-ELONG and GSL-ALK) largely regulate the aliphatic GSL profile in B. oleracea [21]. However, the GSL profile might be altered or manifested by pathogenic infections in plants. Many reports have concluded that resistance to necrotrophs, biotrophs and hemibiotrophs are related to indolic GSLs content in Brassica crops [22]. In kale, two types of aliphatic GSLs (sinigrin and glucoiberin) and one indolic GSL (glucobrassicin) in leaves are modulated upon Xcc infection [23]. In Arabidopsis, GSL is hydrolyzed by a myrosinase gene, PENETRATION2 (PEN2), and accumulated as indolic 4-methoxy-glucobrassicin in cells upon expression of CYP81F2 gene in response against fungal pathogen infection [24].
A number of studies (in vivo and in vitro) have been conducted to evaluate the effects of GSLs and their hydrolyzed products on disease development in Brassica crops. The transgenic A. thaliana with an enhanced amount of individual GSL compound showed resistance against Erwinia carotovora and Pseudomonas syringae pv. maculicola [25]. In B. napus, cultivars with higher GSLs content showed resistance against fungal pathogens (Alternaria spp. and Leptosphaeria maculans) compared to the cultivars with low GSLs [26]. The ITCs are produced upon GSL breakdown and showed biocidal effects in reducing soil-borne plant pathogens [27,28]. GSL-hydrolyzed products (GHP) were found as effective to control several plant pathogenic bacteria, such as Erwinia chrysanthemi, Agrobacterium tumefaciens, Pseudomonas tomato, Pseudomonas cichorii, X. juglandis, X. campestris [11]. Recently, a study on the interaction of kale and Xcc pathogen suggested that indolic GSL compound (glucobrassicin) is more effective in decreasing disease severity than aliphatic GSL compounds [23].
Management of black rot disease is very difficult because of the wide host range of the pathogens. Therefore, secondary metabolites could be used as an alternate biocide for sustainable disease management in Brassica crops [25,29]. To date, only few works have been reported that assess the role of GSLs and their derived products against Xcc pathogen in cabbage. The effects of GSLs, ITCs and plant phenolics have been evaluated on different Brassica species, such as Lepidium sativum, Eruca sativa, B. olerecea var. italica cv. Marathon, B. olerecea var. capitata cv. coracao de boi, B. oleracea var. tronchuda cv. Tronchuda Portuguesa. Positive associations were established among specific GSL components, total GSL content and disease severity in B. rapa against Xcc [11]. This result helps to elucidate the constitutive resistance in plants against Xcc pathogen [17]. Here, we investigated the interaction between GSL profiles of black rot susceptible and resistant cabbage inbred lines, expression profiling of GSL biosynthesis and break-down related genes after infection by Xcc race 4 pathogen. These findings Plants 2020, 9,1121 3 of 16 will be helpful for further research to elucidate the underlying mechanism of host-pathogen interactions at biochemical and molecular levels for developing black rot resistance cabbage cultivars.

Selection of Plant Materials
Two cabbage genotypes SCNU-C-4072 and SCNU-C-3383 were used as source of black rot disease resistance and susceptible lines, respectively, which were selected after screening of 59 inbred cabbage lines collected from the Department of Horticulture, Sunchon National University, Korea against Xcc race 4 pathogen. This screening experiment was conducted in the greenhouse which had a temperature of 28 ± 2 • C, >80% humidity and 16/8 h day/night ( Figure S1 and Table S1). Xcc race 4 (HRI-W-1279A) was collected from Horticulture Research International, Wellesbourne (HRI-W) [30]. This bacterial isolate was maintained on King's B medium and incubated at 30 • C for 48 h [31], from which 1 × 10 8 CFU/mL bacterial isolate was collected and used to infect 35 days old cabbage seedlings following leaf dip method after cutting 1 cm off the tip of the leaves [32]. Three biological replicates were maintained for each cabbage line and high humidity was ensured by covering the infected plants with polythene bags immediately after infection. A set of control and mock (use only water) was also maintained for each cabbage line. Disease symptoms were rated on a 0 to 3 scale after 14 days of inoculation (DAI) [30]

Collection of Leaf Samples for HPLC and PCR
Leaf samples were collected from each of the control, mock-treated and Xcc inoculated plants, at 1, 3 and 5 DAI (Figure 1) for estimating the endogenous GSLs content and expression of GSL biosynthesis and breakdown related genes. Leaf samples were stored immediately at −80 • C after freezing in liquid nitrogen for HPLC (high performance liquid chromatography) and quantitative reverse transcription PCR (qRT-PCR) analyses.

Glucosinolates Identification, Quantification and Analysis
The GSL composition of the cabbage leaf samples was estimated as desulfo-GSL from three biological replicates for each of the control, mock-treated and Xcc infected plants using a modified Plants 2020, 9,1121 4 of 16 HPLC method as previously described [33,34]. Frozen leaf tissue was treated with methanol and stored at −80 • C and then ground to a very fine powder. The powdered leaf samples were stored at 70 • C for 10 min and then brought out at room temperature for 1 h. The samples were centrifuged at 10,000× g at 4 • C for 8 min to remove the undesirable structural components and protein molecules. The supernatant (crude GSL sample) was collected at the end of anion-exchange chromatography. Afterward, the raw GSL sample was desulfurized following the method [33,35] and the GSLs were eluted with 1 mL distilled water. The eluted desulfo-GSLs were purified by high-speed centrifugation at 20,000× g for 4 min at 4 • C followed by filtering through a PTFE (polytetrafluoroethylene) filter (13 mm, 0.2 µm, Advantec, Pleasanton, CA, USA). Purified GSLs were estimated by HPLC on Waters 2695 HPLC system (Waters, Milford, MA, USA) provided with a C18 column (Zorbax Eclipse XBD C18, 4.6 m ×150 mm; Agilent Technologies, Palo Alto, CA, USA). Water and acetonitrile were used as mobile phase solvents. HPLC-mass spectrometry analysis (Agilent 1200 series, Agilent Technologies) was used for identifying individual GSLs [35]. The individual GSL compound was measured at a wavelength of 229 nm using PDA 996 UV-visible detector. A standard curve was prepared with commercial standard sinigrin (SIN) for quantification of the identified individual GSLs.

Expression Analysis of GSL Biosynthesis Pathway and Break-Down Related Genes
A total of 43 genes, of which 38 associated with GSL biosynthesis and 5 GSL breakdown were used for expression analysis. Among them, 11 genes belong to transcription factor (TF) (5 from aliphatic and 6 from indolic groups of GSL biosynthesis pathway). Primers were designed on twenty seven genes, of which 10 were from aliphatic and 17 from indolic GSL biosynthesis pathway ( Figure 2 and Table S2). Primer efficiencies were assessed following the process [33,36] and highly efficient primers were used for expression analysis. The glucosinolate pathway showing the transcription factors, glucosinolate (GSL) biosynthesis and breakdown related genes indicated aliphatic and indolic GSL biosynthesis pathways adopted from previous studies [33,35,37]. A total of 43 genes were selected (15 and 23 genes from aliphatic and indolic GSL biosynthesis pathway and 5 genes from GSL breakdown, respectively). Blue color indicates the genes, which were highly expressed in the resistant line and red color indicates the genes which were highly expressed in susceptible cabbage line after infection with X. campestris pv. campestris race 4. Bold green color number in bracket indicates 1 and 3 days after inoculation.

Total RNA Extraction, cDNA Synthesis and Quantitative Real-Time PCR Analysis
The collected leaf samples were ground in liquid nitrogen and total RNA was extracted from 100 mg leaf tissue using RNeasy mini kit (Qiagen, Valencia, CA, USA). The quantity and quality of RNA were determined using a NanoDrop ND-1000 (260/280 nm) spectrophotometer (NanoDrop, Wilmington, DE, USA) (Supplementary Figure S2) and 1% agarose gel electrophoresis, respectively, prior to use in cDNA (complementary DNA) synthesis for qRT-PCR (Supplementary Figure S3). The cDNA was synthesized using a first-strand cDNA synthesis kit (Thermo Fisher Scientific, MA, USA) following the manufacturer's protocol. The equality of cDNA for each sample was standardized by checking the concentration (60 ng/µL) in NanoDrop as well as comparing the band thickness of end-point PCR product of each sample amplified with actin genes of B. oleracea. Primers were designed on three actin genes named as actin1, actin2, and actin3 with NCBI accessions AF044573 [38], JQ435879 [39] and XM_013753106 [40]. These genes were also used as internal controls for estimating relative gene expression. The qPCR was performed using a 20 µL reaction mixture contained 10 µL 2× Quanti Speed SYBR mix (Thermo Fisher Scientific), 1 µL (10 pmol) each of the forward and reverse gene-specific primers (Supplementary Table S2), 1 µL template cDNA (60 ng) and 7 µL distilled-deionized water. The PCR condition was fixed as initial denaturation at 95 • C for 10 min, 40 cycles of amplification with denaturation at 95 • C for 20 s, annealing at 58 • C for 20 s, and amplification and signal acquisition at 72 • C for 30 s. Each reaction was performed three times as technical replicates using LightCycler96 (Roche, Mannheim, Germany). Livak's comparative 2 -∆∆Ct method [41] was used to calculate the relative expression level of each sample. Average Cq value of three actin genes was used for estimating relative expression of GSL biosynthesis and breakdown related genes.

Statistical Analysis
One-way ANOVA (analysis of variance) and mean separation following Tukey's pairwise comparison for relative expression of each gene and GSL content at different time courses were done using Minitab 18 statistical software (Minitab Inc., State College, PA, USA). Test statistics, degrees of freedom, F-and P-values of statistical significance for GSL content and the relative expression of GSL biosynthetic and breakdown related genes are presented in Tables S3 and S4. A heat map was constructed in Microsoft Excel using conditional formatting options to display the correlation between GSL content and expression values of genes (Tables S5-S9). Principal component analysis (PCA) was done using GSL compounds as determined by HPLC and gene expression values by Minitab 18.

Phenotypic Evaluation of Cabbage Lines Against Black Rot Disease
Fifty nine inbred cabbage lines were screened against black rot disease after infection of Xcc race 4. Most of the lines found as susceptible, whereas lines SCNU-C-060, SCNU-C-064 and SCNU-C-4072 were identified as resistance (Table S1). The resistant lines did not show any disease symptoms on the inoculated leaves up to 14 DAI (days after inoculation); whereas, susceptible lines produced necrotic lesions with characteristic V-shaped symptoms. One resistant (SCNU-C-4072) and one susceptible (SCNU-C-3383) line was used for further study (Figure 1 and Supplementary Figure S1).

Distribution of the GSL Compounds in the Black Rot Resistant and Susceptible Lines
HPLC (high-performance liquid chromatography) was performed for identifying different GSL compounds in the black rot resistant (SCNU-C-4072) and susceptible (SCNU-C-3383) lines ( Figure 3, Supplementary Data S1 and S2). Resistant control plants had higher amount of glucoiberin and progoitrin than susceptible line. Ten GSL compounds (glucoiberin, progoitrin, sinigrin, glucoerucin, gluconapin, glucoiberverin, hydroxyglucobrassicin, glucobrassicin, mythoxyglucobrassicin and neoglucobrassicin) were identified in the resistant line. Whereas 8 GSL compounds were identified in susceptible, except the gluconapin and hydroxyglucobrassicin lines ( Figure 3). Compared to control plants, inoculated resistant and susceptible lines showed significant difference for both aliphatic and indolic GSL content. Aliphatic and indolic GSL compounds were increased significantly in the resistant line in response to Xcc, whereas it was decreased or not induced in the susceptible line. In the resistant line, levels of aliphatic compound (sinigrin and glucoiberverin) were increased significantly by 4.08and 5.19-fold at 3 DAI and glucoerucin was increased 5.44-fold at 1 DAI after Xcc infection compared to mock ( Figure 3, Tables S3 and S5). In the susceptible line, aliphatic compounds (sinigrin, glucoiberverin and glucoerucin) were decreased by 0.68-, 1.22-, and 1.64-fold, respectively, in infected plants compared to mock. The gluconapin level was increased by 4.79-fold at 1 DAI in the resistant line, whereas it was totally absent in the susceptible line ( Figure 3, Tables S3 and S5). The indole GSL compounds (glucobrassicin, mythoxyglucobrassicin, and neoglucobrassicin) were increased by 5.20-fold at 1 DAI as well as 4.69-and 3.61-fold at 3 DAI, respectively, whereas hydroxyglucobrassicin was significantly increased by 10.27-fold at 3 DAI in resistant line. This indolic compound was totally absent in the susceptible line. Glucobrassicin and neoglucobrassicin were decreased by 1.02-and 1.28-fold at 1 DAI and 3 DAI, respectively, in the susceptible line. Mythoxyglucobrassicin was increased by 3.28-fold at 3 DAI in the susceptible line ( Figure 3, Tables S3 and S5). Both aliphatic and indolic GSL compounds were increased in the resistant line upon Xcc infection compared to susceptible line.
Plants 2020, 9, x FOR PEER REVIEW 6 of 17 progoitrin than susceptible line. Ten GSL compounds (glucoiberin, progoitrin, sinigrin, glucoerucin, gluconapin, glucoiberverin, hydroxyglucobrassicin, glucobrassicin, mythoxyglucobrassicin and neoglucobrassicin) were identified in the resistant line. Whereas 8 GSL compounds were identified in susceptible, except the gluconapin and hydroxyglucobrassicin lines ( Figure 3). Compared to control plants, inoculated resistant and susceptible lines showed significant difference for both aliphatic and indolic GSL content. Aliphatic and indolic GSL compounds were increased significantly in the resistant line in response to Xcc, whereas it was decreased or not induced in the susceptible line. In the resistant line, levels of aliphatic compound (sinigrin and glucoiberverin) were increased significantly by 4.08-and 5.19-fold at 3 DAI and glucoerucin was increased 5.44-fold at 1 DAI after Xcc infection compared to mock ( Figure 3, Table S3 and Table S5). In the susceptible line, aliphatic compounds (sinigrin, glucoiberverin and glucoerucin) were decreased by 0.68-, 1.22-, and 1.64-fold, respectively, in infected plants compared to mock. The gluconapin level was increased by 4.79-fold at 1 DAI in the resistant line, whereas it was totally absent in the susceptible line ( Figure 3, Table S3 and Table S5). The indole GSL compounds (glucobrassicin, mythoxyglucobrassicin, and neoglucobrassicin) were increased by 5.20-fold at 1 DAI as well as 4.69-and 3.61-fold at 3 DAI, respectively, whereas hydroxyglucobrassicin was significantly increased by 10.27-fold at 3 DAI in resistant line. This indolic compound was totally absent in the susceptible line. Glucobrassicin and neoglucobrassicin were decreased by 1.02-and 1.28-fold at 1 DAI and 3 DAI, respectively, in the susceptible line. Mythoxyglucobrassicin was increased by 3.28-fold at 3 DAI in the susceptible line ( Figure 3, Table S3 and Table S5). Both aliphatic and indolic GSL compounds were increased in the resistant line upon Xcc infection compared to susceptible line. Vertical bars indicate standard deviation. Different letters indicate statistically significant differences (p < 0.01) between genotypes (R and S lines) and treatment interactions following Tukey test. HPLC-mass spectrometry (HPLC-MS) analysis (using an Agilent 1200 series instrument, Agilent Technologies) was conducted following a previous study [34]. R, resistant; S, susceptible. Vertical bars indicate standard deviation. Different letters indicate statistically significant differences (p < 0.01) between genotypes (R and S lines) and treatment interactions following Tukey test. HPLC-mass spectrometry (HPLC-MS) analysis (using an Agilent 1200 series instrument, Agilent Technologies) was conducted following a previous study [34]. R, resistant; S, susceptible.

Relative Expression of Transcription Factor-and GSL Biosynthesis-Related Genes in Black Rot Resistant Line
We investigated the expression patterns of selected TF (transcription factor)-related genes of the GSL biosynthesis pathway in the resistant line (SCNU-C-4072) by qPCR. Among them, eight genes showed up-regulation upon Xcc infection at different DAI ( Figure 4 and Figure S4). Two genes Plants 2020, 9, 1121 7 of 16 (MYB122-Bol026204 and MYB34-Bol017062) showed higher relative expression by 49.40-and 36.85-fold up-regulation at 1 DAI and 3 DAI, respectively, compared to mock ( Figure 4 and Table S4). Two genes from aliphatic pathway; AOP2-Bo9g006240 and ST5c-Bol030757 showed higher expression by 10.30and 13.12-fold up-regulation at 1 DAI and 3 DAI, respectively, compared to mock ( Figure 4 and Table S7). One indolic biosynthesis gene (CYP81F1-Bol017376) out of four, showed significantly higher expression at 1 DAI by 11.86-fold changed, other three genes, CYP81F2-Bol012237, CYP81F4-Bol032712 and CYP81F4-Bol032714 showed higher expression by 6.40-, 9.69-, and 25.29-fold increased, respectively, at 3 DAI compared to susceptible line and mock ( Figure 4 and Table S8).

Relative Expression of Transcription Factor-and GSL Biosynthesis-Related Genes in Black Rot Resistant Line
We investigated the expression patterns of selected TF (transcription factor)-related genes of the GSL biosynthesis pathway in the resistant line (SCNU-C-4072) by qPCR. Among them, eight genes showed up-regulation upon Xcc infection at different DAI ( Figure 4 and Figure S4). Two genes (MYB122-Bol026204 and MYB34-Bol017062) showed higher relative expression by 49.40-and 36.85fold up-regulation at 1 DAI and 3 DAI, respectively, compared to mock ( Figure 4 and Table S4). Two genes from aliphatic pathway; AOP2-Bo9g006240 and ST5c-Bol030757 showed higher expression by 10.30-and 13.12-fold up-regulation at 1 DAI and 3 DAI, respectively, compared to mock ( Figure 4 and Table S7). One indolic biosynthesis gene (CYP81F1-Bol017376) out of four, showed significantly higher expression at 1 DAI by 11.86-fold changed, other three genes, CYP81F2-Bol012237, CYP81F4-Bol032712 and CYP81F4-Bol032714 showed higher expression by 6.40-, 9.69-, and 25.29-fold increased, respectively, at 3 DAI compared to susceptible line and mock (Figure 4 and Table S8).

Relative Expression of GSL Breakdown Related Genes in Cabbage Lines
The expression of five GSL breakdown related genes showed significant variations for resistant and susceptible lines at different time courses ( Figure 6 and Table S9). Gene PEN2-Bol030092 showed significantly higher expression by 8.47-fold up-regulation at 1 DAI in the resistant line after Xcc infection compared to mock ( Figure 6 and Table S9). Whereas, TGG2-Bol025706 gene showed higher expression by 4.14-fold in the susceptible line at 1 DAI compared to mock. The rest of the genes; TGG1-Bol017328, TGG2-Bol028319 and TGG5-Bol031599, did not show any consistent expression in resistant and susceptible lines ( Figure 6).

Relative Expression of GSL Breakdown Related Genes in Cabbage Lines
The expression of five GSL breakdown related genes showed significant variations for resistant and susceptible lines at different time courses ( Figure 6 and Table S9). Gene PEN2-Bol030092 showed significantly higher expression by 8.47-fold up-regulation at 1 DAI in the resistant line after Xcc infection compared to mock ( Figure 6 and Table S9). Whereas, TGG2-Bol025706 gene showed higher expression by 4.14-fold in the susceptible line at 1 DAI compared to mock. The rest of the genes; TGG1-Bol017328, TGG2-Bol028319 and TGG5-Bol031599, did not show any consistent expression in resistant and susceptible lines ( Figure 6).

Correlation between GSL Components and Expression Level of Genes in the Cabbage Lines
A simple heat map was constructed using expression of TF-related and GSL biosynthesis and breakdown genes and level of individual GSL components after Xcc infection in resistant and susceptible lines ( Figure 7A,B). Pearson's correlation coefficient showed significant positive associations between aliphatic GSL (glucoiberverin and glucoiberin) content and the expression level of ST5c-Bol030757 gene ( Figure 7A). Aliphatic GSL compounds (glucoiberverin, sinigrin, gluconapin and glucoerucin) and the expression of AOP2-Bo9g006240 also showed positive association ( Figure 7A and Supplementary Data S2). Similarly, aliphatic compound progoitrin and GSL biosynthesis related genes (FMOGS-OX5-Bol029100, FMOGS-OX5-Bol031350 and AOP2-Bo3g052110) showed inconsistent positive association. In addition, a highly significant positive correlation was observed between MYB34-Bol017062 and indolic GSL compounds (hydroxyglucobrassicin, glucobrassicin and mythoxyglucobrassicin). Significant positive associations were found between GSL compounds (hydroxyglucobrassicin, mythoxyglucobrassicin and neoglucobrassicin) and indolic GSL genes (MYB122-Bol026204, CYP81F2-Bol012237, CYP81F4-Bol032712 and CYP81F4-Bol032714) ( Figure 7B and Supplementary Data S2).
A principal component analysis (PCA) was done by using total GSL, GSL profiles and expression of GSL biosynthesis and breakdown related genes in two contrasting cabbage lines and identified four principal components (PCs) with an eigen value greater than unity. The first four PCs explained 79.2% of the total variance, of which 32.2%, 21.0%, 14.0% and 12.0% of the variances were included from PC1, PC2, PC3 and PC4, respectively (Table S10). The total variation found in PC1 was manifested by the higher positive coefficients of the TF, GSL biosynthesis and breakdown related genes and higher negative coefficients of GSL profiles and total GSL content in resistance and susceptible lines upon Xcc infection ( Figure 8 and Table S10). PC1 significantly separated as resistant and susceptible groups, which was also confirmed by Tukey's test using overall PCA score ( Figure 8 and Table S10). The highly expressed GSL biosynthetic and breakdown related genes were plotted with total GSL content and most of the GSL constituents in the resistance line, as shown in the PCA biplot ( Figure 8). Results indicate that resistance in cabbage might be attained by synthesizing specific GSL compounds as well as simultaneous actions of breakdown genes.

Correlation Between GSL Components and Expression Level of Genes in the Cabbage Lines
A simple heat map was constructed using expression of TF-related and GSL biosynthesis and breakdown genes and level of individual GSL components after Xcc infection in resistant and susceptible lines (Figures 7A,B). Pearson's correlation coefficient showed significant positive associations between aliphatic GSL (glucoiberverin and glucoiberin) content and the expression level of ST5c-Bol030757 gene ( Figure 7A). Aliphatic GSL compounds (glucoiberverin, sinigrin, gluconapin and glucoerucin) and the expression of AOP2-Bo9g006240 also showed positive association (Figure A principal component analysis (PCA) was done by using total GSL, GSL profiles and expression of GSL biosynthesis and breakdown related genes in two contrasting cabbage lines and identified four principal components (PCs) with an eigen value greater than unity. The first four PCs explained 79.2% of the total variance, of which 32.2%, 21.0%, 14.0% and 12.0% of the variances were included from PC1, PC2, PC3 and PC4, respectively (Table S10). The total variation found in PC1 was manifested by the higher positive coefficients of the TF, GSL biosynthesis and breakdown related genes and higher negative coefficients of GSL profiles and total GSL content in resistance and susceptible lines upon Xcc infection ( Figure 8 and Table S10). PC1 significantly separated as resistant and susceptible groups, which was also confirmed by Tukey's test using overall PCA score ( Figure 8 and Table S10). The highly expressed GSL biosynthetic and breakdown related genes were plotted with total GSL content and most of the GSL constituents in the resistance line, as shown in the PCA biplot ( Figure 8). Results indicate that resistance in cabbage might be attained by synthesizing specific GSL compounds as well as simultaneous actions of breakdown genes.

Black Rot Resistant Cabbage Lines Against Xcc
Black rot disease caused by Xcc is the most devastating in cabbage worldwide, causing significant crop losses [42]. Management of this disease is very difficult because of ever changing races and evolving new races of the Xcc pathogen. Therefore, race-specific resistance in Brassica crops like cabbage is important for marker-assisted breeding. We screened 59 cabbage inbred lines against race 4 of Xcc pathogen and the lines SCNU-C-4072 and SCNU-C-3383 were selected as resistance and susceptible, respectively ( Figure S1 and Table S1).

GSL Compounds Varied in the Leaf Tissues of Resistant and Susceptible Cabbage Lines
We found positive association between disease resistance and GSL accumulation in contrasting cabbage lines upon infection of black rot pathogen. In both of the resistant and susceptible lines, total GSL concentration was increased significantly upon Xcc inoculation at different time points; 1 DAI, 3 DAI and 5 DAI (Figure 3), but amount of GSL content and components differed significantly between resistance and susceptible lines. Total GSL level was increased in leaves of B. rapa resistance cultivar upon infection of Leptosphaeria maculans and Fusarium oxysporum; whereas GSL was decreased in the susceptible line [43]. Recently, an increased level of GSL content was reported in resistance cabbage lines against ring spot disease [34]. A similar result was also found in the susceptible B. napus cultivars infected with Alternaria brassicae pathogen [43,44]. In a previous study, a positive association was found between pathogen-induced indolic GSL compounds and infection of Sclerotinia sclerotiorum [45]. In the case of plant pathogenic bacteria, myrosinase enzyme mediated GSL hydrolyzed products to act as tolerant against Xcc [11,17] Aliphatic GSL (sinigrin, gluconapin, glucoiberverin and glucoerucin) and indolic (glucobrassicin, hydroxyglucobrassicin, mythoxyglucobrassicin and neoglucobrassicin) content was found to increase due to Xcc infection in resistant cabbage line ( Figure 3) and represents its involvement in improving resistance against Xcc pathogen in cabbage. Aliphatic GSL (glucoiberverin, indolic; glucobrassicin and mythoxyglucobrassicin) was found to increase in cabbage lines resistant to Mycosphaerella brassicicola [34]. The aliphatic GSL compound, sinigrin showed a potential role in inhibiting the growth of S. sclerotiorum in kale and Arabidopsis [23,46]. It was reported that the aliphatic gluconapin can defend B. rapa plants against Xcc pathogen [17], thereby a higher amount of gluconapin and hydroxyglucobrassicin was found in resistant B. rapa cultivar [43]. In kale, higher hydroxyglucobrassicin exhibited resistant to the Xcc pathogen [46]. A study on kale and Xcc pathogen interactions suggested that the indolic GSLs compound (glucobrassicin) showed as more effective than aliphatic GSLs in disease resistance [23]. Therefore, it could be concluded that increased level of aliphatic and indolic compounds may confer resistance against Xcc pathogen in cabbage.

Black Rot Pathogen Induced GSL Biosynthesis Genes in Contrasting Cabbage Lines
The qPCR profiles showed differential gene expression in the black rot resistant and susceptible cabbage lines (Figures 2 and 4-6). Eight genes showed consistent higher relative expression level in the resistant cabbage line (Figures 2 and 4). Among them, two TF, 2 aliphatic GSL and four genes were involved in indolic GSL biosynthesis. Similarly, enhanced expression was found in MYB34-Bol017062, MYB122-Bol026204 and ST5c-Bol030757 genes in the resistant cabbage line, which conferred resistance to ring spot and white mold diseases, respectively [34,36].

Association of Aliphatic GSL Genes with Individual GSL Compound in Black Rot Resistance
Up-regulation of ST5c-Bol030757 and AOP2-Bo9g006240 genes led to increase aliphatic GSL content in the black rot resistant line upon Xcc infection. Increased expression of aliphatic GSL biosynthesis genes, ST5b and ST5c were found to be involved directly in alteration of desulfoglucosinolates to glucoiberin and glucoiberverin [47]. In this study, increased expression of ST5c-Bol030757 was associated with higher levels of glucoiberverin content in resistant line ( Figure 7A and Supplementary data S2) supported by the reports [33,36]. Moreover, AOP2 gene converts methylsulphinylalkyl glucosinolates to sinigrin and gluconapin [47]. In B. rapa, overexpression of AOP2 gene catalyzes conversion of beneficial glucoraphanin to gluconapin [37]. In Arabidopsis, AOP2 gives positive feed-back in regulation of controlling glucosinolate biosynthesis by optimizing the resources in defensive metabolites [48]. AOP2 gene expression showed positive relation between up-regulation of the gene and increased glucoerucin content in cabbage inbred lines [33]. Our results also suggested that Xcc induced up-regulation of genes and increased level of aliphatic compounds may have a link to black rot resistance in cabbage ( Figure 7A and Supplementary data S2). However, detailed molecular studies are needed for precision of this association either by gene editing or knock-out mutation of the particular gene.

Association of Indolic GSL Genes and Individual GSL Compounds in Black rot Resistance
In this study, two TF (MYB122-Bol026204 and MYB34-Bol017062) and four indolic GSL biosynthesis genes (CYP81F1-Bol017376, CYP81F2-Bol012237, CYP81F4-Bol032712 and CYP81F4-Bol032714) were increased significantly in the resistant line upon Xcc infection, which lead to an increase in the indolic compounds (glucobrassicin, hydroxyglucobrassicin, methoxyglucobrassicin and neoglucobrassicin) ( Figure 7B and Supplementary data S2). The MYB34 gene regulates indolic GSL biosynthesis directly in Arabidopsis [49] and in B. oleracea [33,35]. MYB34 together with MYB51 and MYB122 regulated resistance in Arabidopsis after Plectosphaerella cucumerina infection and the indolic GSL biosynthesis genes were triggered by GSL breakdown products induced by PEN2 gene [50]. Accumulation of indolic GSL compound tends to up-regulate CYP81F1, CYP81F2, and CYP81F4 genes in the resistant line (Figures 2, 3 and 7B), which is supported by the association between expression of CYP81F1-Bol017376, CYP81F2-Bol012237 genes and glucobrassicin content in B. oleracea [34,50]. Methoxyglucobrassicin level was increased up to 30-47% in response to blackleg pathogen in B. napus after 5-8 days of inoculation [51]. Anti-fungal activity of methoxyglucobrassicin, glucobrassicin and sinigrin was reported in an in vitro analysis [52]. In this study, the indolic GSLs (glucobrassicin, methoxyglucobrassicin, hydroxyglucobrassicin and neoglucobrassicin) were found to increase in resistant line upon Xcc infection ( Figure 7B and Supplementary data S2), suggesting their role in conferring resistance against Xcc pathogen in cabbage. Since, CYP81F1-Bol017376, CYP81F2-Bol012237, CYP81F4-Bol032712 and CYP81F4-Bol032714 genes were involved in methoxylation and conversion of glucobrassicin to 4-methoxyglucobrassicin, 4-hydroxyglucobrassicin and neoglucobrassicin, which may boost-up resistance in cabbage. Sixteen GSL biosynthesis genes were highly up-regulated in susceptible line (Figures 2 and 5) and may be involved in the susceptibility to Xcc in cabbage. These results are consistent with low accumulation of individual GSL compound and lower expression of GSL biosynthesis genes in cabbage lines and vice-versa [33].

Expression of GSL Breakdown Related Genes in Cabbage Lines after Xcc Inoculation
Glucosinolate breakdown related gene (PEN2-Bol030092) showed significant up-regulation in black rot resistant line at 1 DAI (Figures 2 and 6, and Table S7). A previous study reported that PEN2 gene is involved in resistance in Arabidopsis via callose deposition and glucosinolate stimulation [53]. Moreover, accumulation of indolic GSL compound in Arabidopsis after fungal infection is triggered by the myrosinase PEN2 gene [54]. PEN2 plays a key role in triggering the expression of indolic GSL biosynthesis genes in response to necrotrophic fungal pathogen [50]. The GSL hydrolysis products induced by myrosinase were reported to act as tolerance against Xcc [11,17]. The GSL-myrosinase defense system could be altered by the expression of GSL biosynthesis and breakdown related genes and enhance resistance to herbivores [54]. Our PCA results also confirmed the relations of susceptibility and resistance reactions with different GLS compounds and up-regulation of the respective gene at particular time courses.

Conclusions
A positive association between expression of GSL biosynthesis and breakdown related genes and individual GSL compound was found in resistant and susceptible cabbage lines upon infection by Xcc race 4. Aliphatic (glucoiberverin, sinigrin, gluconapin and glucoerucin) and indolic (glucobrassicin, hydroxyglucobrassicin, methoxyglucobrassicin and neoglucobrassicin) compounds were associated positively with black rot resistance in cabbage. Individual GSL compounds and their associated genes could be putative candidates for biochemical and genetic stimuli of black rot resistance in cabbage. The predicted candidate genes could be further confirmed by functional characterization to improve black rot resistance breeding in cabbage.