Surface-sterilized seeds of Brassica napus cultivars (cv. Capitol and Mosa) were sown in bed soil in a tray and grown in the greenhouse at 24°C with 65% relative humidity in a 1.67 L plastic pot. Natural light was supplied with metal halide lamps that generated c. 400 μmol photons m⁻² s⁻¹ at the canopy height for 16 h day⁻¹. At the four-leaf stage, morphologically similar plants of each cultivar (e.g., 24 plants per cultivar) were divided into two groups for the control (non-pathogen inoculated) and the pathogen-inoculated groups. The pathogenic bacterial (Xcc) strain (KACC No-10377) was collected from the Korean Agricultural Culture Collection. Bacterium inoculum was cultured in a Yeast Dextrose Calcium Carbonate (YDC) agar plate for 48 h at 30°C and adjusted to a concentration of 10⁸ CFU/ml (0.2 OD A600 nm) with 0.85% NaCl solution. The four youngest fully expanded leaves from each plant were inoculated by clipping the leaf edges near the veins using mouth tooth forceps dipped into the bacterial suspension for every inoculation. The experiment was conducted by a completely randomized design with three biological replications. The leaves of two group plants were collected at 0, 3, 9, and 15 days post-Xcc-inoculation (DPI). For the sampling of Xcc-symptom leaves, non-living dried tissues were removed. The collected leaf samples were immediately frozen in liquid nitrogen and stored in a deep freezer (−80°C) for further analysis.

The H₂O₂ level was determined according to Lin and Kao (2001). Approximately, 200 mg of fresh leaves was extracted with 1.5 ml of 50 mM phosphate buffer (pH 6.8) and then centrifuged at 6,000 × g for 25 min. After centrifugation, 3 ml of extract solution was mixed with 0.5 ml of 0.1% titanium chloride in 20% (v/v) H₂SO₄ and then centrifuged at 10,000 × g for 5 min. The absorbance was immediately determined at 410 nm and calculated using the coefficient of absorbance 0.28 μM⁻¹ cm⁻¹.

Quantitative analysis of phytohormones in the leaf tissue was performed through high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) (Pan et al., 2010). Notably, 50 mg of the ground fresh leaf sample were extracted with 500 μl of extraction solvent and 2-propanol/H₂O/concentrated HCl (2:1:0.002, v/v/v); 1 ml of dichloromethane was added to the supernatant, which was centrifuged at 13,000 × g for 5 min at 4°C. The lower phase was poured into a clean screw-cap glass vial, dried in nitrogen, and dissolved in pure methanol. After proper vertexing and sonicating, the completely dissolved extract was moved to a reduced volume liquid chromatography vial. The hormones, namely, SA, JA, and ABA, were analyzed by a reversed-phase C18 Gemini high-performance liquid chromatography (HPLC) column for HPLC-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) analysis. Agilent 1100 HPLC (Agilent Technologies), Waters C18 column (150 × 2.1 mm, 5 μm), and API3000 MSMRM (Applied Biosystems) were used for this analysis.

Endogenous proline and P5C levels in the leaf tissue were determined according to La et al. (2020). Approximately, 200 mg of fresh leaves were homogenized in 3% of sulfosalicylic acid and that was centrifuged at 13,000 × g for 10 min. The collected supernatant was used for the proline and P5C analysis. The resulting supernatants were mixed with ninhydrin solution containing acetic acid and 6 M H₃PO₄ (v/v, 3:2) and boiled for 1 h at 100°C. Toluene was added to the solution, which was incubated at room temperature for 30 min. The absorbance was measured at 520 nm and calculated using l-proline as the standard.

For P5C analysis, the collected supernatants were mixed with 10 mM of 2-aminobenzaldehyde dissolved in 40% of ethanol and incubated at 37°C for 2 h until yellow color appeared (La et al., 2020). The absorbance was measured at 440 nm and calculated using an extinction coefficient of 2.58 mM⁻¹ cm⁻¹.

Total RNA was isolated from 200 mg of fresh ground leaf tissue using RNAiso Plus (Takara, Kusatsu, Japan). Reverse transcription of 2 μg RNA from each sample into complementary DNA (cDNA) was performed using the GoScript Reverse Transcription System (Promega). Quantitative RT-PCR analysis was performed using a Bio-Rad CFX96 qRT-PCR detection system (Bio-Rad), SYBR Premix Ex Taq (TaKaRa, Dalian, China), and specific primers (Supplementary Table 1). Actin was used to normalize the gene expression level and quantified using the 2^ΔΔCt method (Livak and Schmittgen, 2001). The expression levels of the target genes were quantified in duplicates for the three biological replications of each treatment.

The experiment was conducted using a completely randomized design with three biological replications for four sampling dates, two Xcc-treatments, and two B. napus cultivars. Duncan's multiple range test was used to compare the means of separate replicates for each sampling time. The differences were considered statistically significant at p < 0.05. All statistical analyses were performed using SAS 9.1.3 (SAS Institute Inc., Cary, NC, USA).

In the Xcc-inoculated leaves, visibly distinct differences in disease development were observed between the two examined cultivars; onset of yellowing (from 3 DPI) and V-shaped necrotic lesions (at 3–15 DPI) occurred earlier in cv. Mosa than in cv. Capitol. Additionally, the lesions were much larger in cv. Mosa than in cv. Capitol (Figures 1A,B). In the susceptible cultivar (Mosa), Xcc-induced disease symptoms were distinguished in two phases characterized by minor yellowing up to 3 DPI and severe necrosis development during 9–15 DPI (Figure 1B).

In both cultivars, Xcc inoculation significantly enhanced H₂O₂ concentration throughout the experimental period. However, Xcc-responsive increases in the H₂O₂ level were relatively higher in cv. Mosa, especially after 9 DPI. The highest concentration of H₂O₂ in cv. Capitol and cv. Mosa was recorded at 15 DPI (4.0 nmol g⁻¹ FW; Figure 1C) and 9 DPI (6.5 nmol g⁻¹ FW; Figure 1D), respectively. These results suggest that higher Xcc-responsive H₂O₂ accumulation in cv. Mosa might reflect disease symptom development.

Botrytis-induced kinase-1, a receptor-like cytoplasmic-kinase that is required to activate PTI, was significantly (p < 0.001) enhanced at only 3 DPI in both cultivars and was upregulated much more in cv. Capitol (Figures 2A,B). The expression level of ZAR1, a CC-NB-LRR type R-gene, was enhanced significantly from 9 DPI to a maximum level at 15 DPI (4.0-fold) in cv. Capitol (Figure 2C) but was depressed or unchanged in cv. Mosa (Figure 2D). TAO1, a TIR-NB-LRR type R-gene, was upregulated from 9 DPI only in cv. Mosa (Figures 2E,F). In summary, strong expression of BIK1 in cv. Capitol at the early phase (3 DPI) led to a consecutive enhancement in ZAR1-expression at the later phase (9–15 DPI) as an immunity response. Less expressed-BIK1 at the early phase (3 DPI) in cv. Mosa might be responsible for TAO1 expression to induce susceptibility at the later phase (9–15 DPI).

Xanthomonas campestris pv. campestris inoculation significantly enhanced the endogenous SA concentration in both cultivars. The Xcc-induced SA enhancement was distinct at 15 DPI (+5.97-fold) in cv. Capitol (Figure 3A) and much higher in cv. Mosa, with estimated 17.30-fold and 9.74-fold increases at 9 and 15 DPI, respectively, compared with the control (Figure 3B). Xcc inoculation also significantly increased JA concentration in both cultivars. However, the Xcc-induced JA pattern was distinctly different, with a maximum concentration at 15 DPI (2.68 ng g⁻¹ FW, with a progressive Xcc-induced enhancement) in cv. Capitol (Figure 3C) and maximum concentration at 3 DPI (4.34 ng g⁻¹ FW, with a progressive decrease in Xcc-responsive enhancement) in cv. Mosa (Figure 3D). A significant decrease in the Xcc-responsive ABA level was observed only at 15 DPI in cv. Capitol (Figure 3E). However, in cv. Mosa, the Xcc-responsive increase in the ABA level was prominent from 9 DPI (Figure 3F). The resulting SA/JA ratio gradually increased to a maximum (17.1) at 15 DPI in cv. Capitol (Figure 3G); the SA/JA ratio increased more significantly in cv. Mosa (especially during 9–15 DPI) from ~27.07 to 39.36 (Figure 3H).

ICS1, an SA-synthesis-related gene, was upregulated by Xcc inoculation in both cultivars. Xcc-enhanced ICS1 expression was relatively higher in cv. Mosa, especially from 9 DPI (Figures 4A,B). The SA-signaling-related gene PR1 showed a similar pattern with ICS1, with the highest expression level at 15 DPI (3.98-fold) in cv. Capitol and 5.54-fold in cv. Mosa (Figures 4C,D). The ABA synthesis-related gene NCED3 and the ABA signaling gene MYC2 were significantly enhanced only in cv. Mosa from 9 DPI (Figures 4E–H). LOX2, a JA-synthesis-related gene, was significantly upregulated from 9 DPI and reached the highest expression level at 15 DPI (2.0-fold) in cv. Capitol; LOX2 expression in cv. Mosa was the highest at 3 DPI (2.27-fold) and gradually decreased until 15 DPI (Figures 4I,J). Xcc-responsive enhancement of the JA-signaling-related gene PDF1.2 increased during 9–15 DPI in cv. Capitol and gradually decreased from 3 DPI (the highest expression level of 3.23-fold) to 15 DPI (1.67-fold) in cv. Mosa (Figures 4K,L). In summary, it was shown that higher enhancement in SA- and ABA-responses in cv. Mosa, whereas the progressive increase in Xcc-enhanced JA-responses in cv. Capitol.

The Xcc-responsive P5C expression and proline pattern were noticeably different in the two cultivars (Figure 5). Xcc inoculation enhanced P5C only at 3 DPI (0.54 μmol g⁻¹ FW) in cv. Capitol, whereas the Xcc-responsive increase in P5C was significant from 3 DPI and gradually increased up to 15 DPI (0.66 μmol g⁻¹ FW) in cv. Mosa (Figures 5A,B). The proline content increased significantly with Xcc inoculation only at 3 DPI (62.66 μg g⁻¹ FW) in cv. Capitol, whereas Xcc-responsive proline accumulation occurred from 3 DPI and recorded the highest level (84.79 μg g⁻¹ FW) at 9 DPI in cv. Mosa (Figures 5C,D). The expression of P5CS2, a P5C synthesis-gene, was upregulated by Xcc inoculation only at 3 DPI in cv. Capitol (Figure 6A). Xcc-responsive enhancement of P5CS2 expression gradually increased and reached the highest expression level at 15 DPI (4.9-fold) in cv. Mosa (Figure 6B). The expression of P5CR, an encoding gene of P5C reductase that catalyzes the reduction of P5C to proline, was parallel to that of Xcc-responsive P5CS2 in both cultivars (Figures 6C,D). The proline degradation-related gene PDH was induced by Xcc infection in both cultivars and resulted in the highest expression level at 15 DPI, as estimated by 4.4-fold and 5.31-fold increases in cv. Capitol (Figure 6E) and cv. Mosa (Figure 6F), respectively. The results indicated that a distinct increase in P5C and proline at the later phase in cv. Mosa might be closely associated with disease susceptibility.

Black rot disease of oilseed rape (Brassica napus) caused by Xcc is a major threat that reduces the quality and productivity of Brassicaceae crops (Jensen et al., 2005; Vicente and Holub, 2012). Xcc-induced disease susceptibility is distinguished by necrotic lesions in the pathogen-inoculated leaves and concurrently characterized based on the higher increases in reactive oxygen species (ROS). In this study, distinct differences in the appearances of yellowing and V-shaped necrosis development in the Xcc-inoculated leaves were observed. At 15 DPI, much larger necrotic lesions with 1.35-fold higher H₂O₂ concentration in cv. Mosa were observed compared with those estimated in cv. Capitol (Figure 1); thus, “Mosa” and “Capitol” were designated as susceptible and resistant cultivars, respectively. These designations were confirmed by our previous study, which directly elucidated cultivar variation in disease susceptibility and disease responses by estimating redox status and phenylpropanoid synthesis in relation to hormonal status in the B. napus-Xcc pathosystem with six cultivars (Islam et al., 2017). The Xcc-responsive increase in H₂O₂ concentration, which might reflect the severity of disease symptoms, was found to be involved in R-gene expression (Mamun et al., 2020). Xcc-induced disease symptoms developed in two distinct phases (i.e., “early”, a minor yellowing up to 3 DPI and “later,” a severe necrosis development during 9–15 DPI) (Figures 1A,B). In this context, the cultivar variation in the Xcc-responsive immune-related genes, the hormone status, and the signaling and proline metabolism in two different phases were interpreted with relation to PTI and ETI or ETS.

In the pathogen-infected plants, the first layer of innate immune is initiated by the perception of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) to induce PTI (Zipfel, 2014; Bigeard et al., 2015). The second layer is a long-lasting defense response (ETI), which has been known to be activated by interactions between the pathogen effector and plant R-genes (Jones and Dangl, 2006; Joshi and Nayak, 2011; Cook et al., 2015). The perception of PAMPs by PRRs activates the cytoplasmic protein kinase BIK1, which simultaneously activates a calcium channel and phosphorylation of respiratory burst oxidase homolog (RBOH) (Kadota et al., 2014), which is critical for PAMP-induced ROS generation (Suzuki et al., 2011; Macho et al., 2012). In this study, BIK1 expression was significantly (p < 0.001) enhanced at 3 DPI (Figures 2A,B), with a concurrent increase in H₂O₂ concentration (Figures 1C,D) in both cultivars. Xcc-responsive enhancement of BIK1 expression was much higher in cv. Capitol (+6.3-fold) than in cv. Mosa (+2.6-fold) (Figures 2A,B). BIK1 is required to activate PTI against several necrotrophic pathogens (Veronese et al., 2006; Zhang et al., 2010) and could possibly regulate pattern-triggered responses, e.g., ROS (especially H₂O₂) production (Zhang et al., 2010). However, the Xcc-responsive pattern between BIK1 expression (Figures 2A,B) and H₂O₂ concentration (Figures 1C,D) at 3 DPI was not consistent in the two cultivars, indicating that BIK1 expression might be multidimensional with another regulatory signaling (e.g., hormonal) rather than a single expression with ROS.

In fact, at 3 DPI, the Xcc-responsive increase in the SA level was at a similar rate in both cultivars (Figures 3A,B) but the Xcc-responsive increase in the JA level was significant only in cv. Mosa (Figure 3D). The increase in the SA level is widely observed under drought stress (La et al., 2019; Park et al., 2021) and in pathogen-infected leaves (Islam et al., 2017; Mamun et al., 2020) and is involved in the regulation of stress responses. Interestingly, Veronese et al. (2006) reported that BIK1 functions as a positive regulator of resistance to fungal disease (Botrytis cinerea) and as a negative regulator to bacterial disease (Pseudomonas syringae) at normal SA levels. However, for SA above a certain threshold, BIK1 triggers the suppression of mechanisms required for resistance to Botrytis, while still promoting mechanisms for resistance to Pseudomonas. The evidence of the SA interaction with other hormones (especially with JA and ABA) in pathogen defense responses has been proven (Veronese et al., 2006; Islam et al., 2017; Mamun et al., 2020). Given the asymptomatic or negligible Xcc-responsible symptoms in both cultivars in the early phase (up to 3 DPI) (Figures 1A,B), Xcc-responsive enhancement BIK1 expression and SA and/or JA status in the two cultivars at this phase reflect the PTI process, which inoculation induced differently in resistant (cv. Capitol) and susceptible (cv. Mosa) by Xcc-infection. Therefore, BIK1-mediated alteration in the hormonal status in the early phase would be a PTI activation that further regulates the resistant or susceptible interaction (Veronese et al., 2006).

Effector-triggered immunity is a long-lasting defense response, which is activated through interaction between R-genes and bacterial effectors (Joshi and Nayak, 2011; Wu et al., 2014). Two major types of R-genes [i.e., coiled-coil-nucleotide binding site-leucine-rich repeat (CC-NB-LRR)-type (e.g., ZAR1) and toll/interleukin-1 receptor-nucleotide binding site-leucine-rich repeat (TIR-NB-LRR)-type (e.g., TAO1)] regulate the activation of the plant immune response to counteract pathogenic infection (Jones and Dangl, 2006; Joshi and Nayak, 2011; Wang et al., 2015). In this study, ZAR1 was significantly upregulated from 9 DPI during the later phase (9–15 DPI) and reached the highest value at 15 DPI in the resistant cultivar “Capitol” (Figure 2C), in which Xcc-responsive BIK1 upregulation (+6.3-fold) was distinct at 3 DPI (Figure 2A). The Xcc-responsive pattern of ZAR1 expression in cv. Capitol was consistent with the JA level (Figure 3C) and was accompanied by the enhanced JA synthesis-related gene LOX2 (Figure 4I) and the JA signaling gene PDF1.2 (Figure 4K). However, in the susceptible cultivar “Mosa,” Xcc-responsive upregulation of TAO1 was significant during the later phase (9–15 DPI) (Figure 2F). In cv. Mosa, Xcc-responsive enhancement of TAO1 expression during the later phase was consistent with the SA level pattern (Figure 3B), the SA synthesis gene ICS1 (Figure 4B), and the signaling gene PR1 (Figure 4D). Similarly, the Xcc-responsive ABA level (Figure 3F), the ABA synthesis gene NCED3 (Figure 4F), and the ABA signaling gene MYC2 (Figure 4H) gradually increased in a TAO1-dependent manner. However, the Xcc-responsive JA level (Figure 3D), the JA synthesis gene LOX2 (Figure 4J), and the JA signaling gene PDF1.2 (Figure 4L) decreased antagonistically with increasing SA and ABA responses during the later phase. These results indicate that the Xcc-induced TAO1-dependent activation of SA and JA signaling antagonistically depressed JA signaling and mediates the ETS during the later phase. Similar results have been reported that TIR-NB-LRR-type R-gene is involved in the regulation of ETS through SA signaling (Lorang et al., 2007; Mamun et al., 2020). In addition, the present data and previous data suggest that the Xcc-responsive TAO1-dependent SA level and signaling were antagonistic interactions with JA responses and synergistic with ABA signaling in the susceptible cultivar “Mosa” as an ETS response (Veronese et al., 2006; Mamun et al., 2020). In addition, we found the different patterns of Xcc-responsive SA and JA, which mainly attributed to different severities of disease between the previous (Islam et al., 2017) and this study. In fact, the infection and disease symptom development were much rapid in cv. Mosa compared with those in cv. Capitol. Although the discrepancies exist in the individual hormone level, the Xcc-responsive SA/JA ratio pattern was found to be the same in both experiments. For instance, a much higher SA/JA ratio was observed at the later necrotrophic phases (9 DPI and 15 DPI) in the susceptible response (cv. Mosa), which is in line with the previous findings.

Therefore, in our previous study with six B. napus cultivars, the susceptibility to Xcc was characterized by the enhanced alteration of the SA/JA ratio as a negative regulator of redox status and phenylpropanoid synthesis (Islam et al., 2017), in accordance with the higher enhancement in cv. Mosa (Figure 3H). Furthermore, free proline accumulates in response to a wide range of abiotic and biotic stresses (Rejeb et al., 2014; Qamar et al., 2015; La et al., 2019). In the later phase (9–15 DPI) of this study, Xcc inoculation did not affect P5C and the proline level in cv. Capitol (Figures 5A,C), whereas the Xcc-responsive increase in these compounds was highly significant (p < 0.001) (Figures 5B,D) and concurrent with the enhanced expression of proline synthesis-related genes (P5CS2 and P5CR) in cv. Mosa (Figures 6B,D). In addition, during the later phase, Xcc-responsive enhancement of the proline level and its synthesis-related genes in the susceptible cultivar “Mosa” was found to be followed by the TAO1-mediated SA- and ABA-dependent manner only. The SA-mediated H₂O₂ signaling from NADPH-oxidase initiates proline accumulation under pathogen or drought stress (Szabados and Savoure, 2009; La et al., 2019). Proline accumulation is partially regulated by an ABA-dependent signaling pathway (Savouré et al., 1997; Verslues and Sharma, 2010). Furthermore, the proline degradation-related gene PDH was highly enhanced in both cultivars (Figures 6E,F). Proline degradation by proline dehydrogenase is required for the development of hypersensitive responses during pathogenesis (Cecchini et al., 2011; Senthil-Kumar and Mysore, 2012). Several studies have suggested the presence of excessive levels of proline under stressful conditions due to a more active proline/P5C cycle. Thus, more electrons are delivered to the mitochondrial electron transport chain, leading to ROS production and the generation of hypersensitive responses (Verslues and Sharma, 2010; Rejeb et al., 2014). The data obtained during the later phase indicate that the activated interaction between ZAR1 and JA signaling restricts proline accumulation by activating proline degradation and leading to moderate disease symptoms as an ETI response in cv. Capitol; additionally, the interaction of TAO1 with SA and ABA signaling enhanced proline accumulation by activating proline synthesis and leading to severe susceptibility as ETS in cv. Mosa.