Light- and Relative Humidity-Regulated Hypersensitive Cell Death and Plant Immunity in Chinese Cabbage Leaves by a Non-adapted Bacteria Xanthomonas campestris pv. vesicatoria

Article information

Plant Pathol J. 2024;40(4):358-376

Publication date (electronic) : 2024 August 1

doi : https://doi.org/10.5423/PPJ.OA.03.2024.0057

Young Hee Lee^1,2, Yun-Hee Kim³, Jeum Kyu Hong^1,2,

¹Laboratory of Horticultural Crop Protection, Division of Horticultural Science, Gyeongsang National University, Jinju 52725, Korea

²Agri-Food Bio Convergence Institute, Gyeongsang National University, Jinju 52725, Korea

³Laboratory of Plant Molecular Physiology, Department of Biology Education, Gyeongsang National University, Jinju 52828, Korea

^*Corresponding author. Phone) +82-55-772-3258, FAX) +82-55-772-3257, E-mail) jkhong@gnu.ac.kr

Handling Editor : Cecile Segonzac

Received 2024 March 26; Revised 2024 June 10; Accepted 2024 July 8.

Abstract

Materials and Methods

RNA isolation and quantitative RT-PCR analyses

Total RNA was isolated from the leaves using RiboEx solution (GeneAll, Seoul, Korea) (Lee et al., 2022b). Total RNAs prepared in DEPC-treated water were treated with RNase-free DNase (Promega, Madison, WI, USA) to eliminate any contaminated genomic DNA according to the manufacturer’s protocol. cDNAs were synthesized from the total RNA using SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). Quantitative real-time PCR analysis was performed using a Bio-Rad CFX96 thermal cycler (Bio-Rad, Hercules, CA, USA) with EvaGreen fluorescent dye (Biotium, Inc., Fremont, CA, USA). Linear data were normalized to the mean threshold cycle of Brassica rapa actin 7 gene (BrAct7). Gene-specific PCR primer pairs are listed in Table 1.

Table 1

Nucleotide sequences of oligonucleotide primers of Chinese cabbage genes used for the quantitative RT-PCR in this study

Results

Different plant cell death and in planta bacterial growth in the Xcc- and Xcv-inoculated Chinese cabbage leaves

Inoculating adapted and non-adapted strains of X. campestris led to different cell death responses in the Chinese cabbage leaves (Fig. 1). The Xcc- and Xcv-inoculated leaves were observed at 0 (immediately after the bacterial inoculation) and 24 h (Fig. 1A). Xcc did not cause any visual tissue collapse in the inoculated leaves, but Xcv triggered rapid tissue damage at the inoculated site at 24 h. The different responses of the inoculated leaves were clearly shown by removing chlorophylls in ethanol. In particular, brown tissue discoloration was only found in the Xcv-inoculated leaf areas at 24 h but not in mock- and Xcc-inoculated ones.

Fig. 1

Non-adapted bacteria-triggered hypersensitive cell death in Chinese cabbage leaves. (A) Differential tissue damage in the leaves inoculated by bacterial suspensions (10⁷ cfu/ml) of the adapted and non-adapted strains of Xanthomonas campestris pathovars. A virulent adapted strain 8004 of X. campestris pv. campestris (Xcc) and a non-adapted strain Bv5-4a.1 of X. campestris pv. vesicatoria (Xcv) were syringe-infiltrated into the abaxial surface of the primary leaves of Chinese cabbage seedlings. Localized tissue collapse in the Xcv-inoculated leaves were visualized with or without removing chlorophylls by ethanol. Photos were taken at 0 (immediately after the bacterial inoculation) and 24 h. (B) Bacterial growth of Xcc and Xcv in the inoculated leaves. Xcc and Xcv bacterial suspensions (10⁷ cfu/ml) were syringe-infiltrated, and bacterial numbers were counted at 0, 6, and 24 h. The data are the means ± standard errors of the five independent experimental replicates. Each experiment contained four biological replicates. Means with the same letter above the bars are not significantly different at the 5% level by the least significant difference tests. (C) Microscopic observations of different cell death occurrences in the leaf tissues inoculated by Xcc and Xcv at 24 h. Dying leaf cells stained by trypan blue in the leaf tissues were observed under a light microscope. Scale bars = 100 μm. (D) Trypan blue-stained cells in the inoculated leaves seen in the photos were counted and quantified as numbers/mm². The data are the means ± standard errors of the five independent experimental replicates. Four to 12 photos for each treatment were used for an independent experiment. Means with the same letter above the bars are not significantly different at the 5% level by the least significant difference tests. (E) Mesophyll cells of the Xcc and Xcv-inoculated leaves at 24 h. Arrows indicate collapsed leaf cells. Scale bars = 25 μm.

To investigate bacterial proliferation within the inoculated leaf tissues in situ during the different cell death responses caused by adapted and non-adapted strains of X. campestris, high doses (10⁷ cfu/ml) of the bacterial suspensions were inoculated and living bacterial cell numbers were counted at 0, 6 and 24 h (Fig. 1B). Xcc growth decreased temporarily at 6 h (0.96 log-fold) compared to 0 h but increased at 24 h (1.09 log-fold) compared to the growth at 0 h. In contrast, bacterial growth for the Xcv was found differently at the same time points. Xcv growth increased at 6 h compared to 0 h (1.05 log-fold) but returned to the initial level of bacterial number at 24 h. Xcv grew slightly more than Xcc at 6 h (1.11 log-fold). But Xcc growth was significantly higher (1.10 log-fold) than Xcv at 24 h, at which the hypersensitive response was found in the leaves inoculated by Xcv.

Plant cell death occurrence was also predominantly observed in the leaves inoculated by the non-adapted bacteria under a light microscope (Fig. 1C). Under observation at 100× magnification, no distinct trypan blue staining was found in the mock-inoculated leaves and only scattered blue stains were observed in the Xcc-inoculated leaves at 24 h. But non-adapted Xcv inoculation led to prominent trypan blue stains widespread in the inoculated leaves at 24 h. The number of trypan blue-stained cells in the inoculated leaves was measured (Fig. 1D). Xcc inoculation increased the number of trypan blue-stained cells compared to mock inoculation, and Xcv inoculation led to a higher number of cells stained by trypan blue. The inoculated leaf tissues were observed under observation at 400× magnification (Fig. 1E). Most mesophyll cells showed typical round shapes in the mock-inoculated leaves. A few collapsed and stained cells were observed in the Xcc-inoculated leaves, and most cells without distinct trypan blue staining looked normal in cellular shape. Many mesophyll cells were collapsed and stained in the Xcv Bv5-4a.1-inoculated leaves, and most cells without deep staining also showed degenerated forms.

HCD-associated electrolyte leakage and lipid peroxidation in Chinese cabbage leaves by non-adapted Xcv

Electrolyte leakage and lipid peroxidation were differently found in the Chinese cabbage leaves during different cell death responses to adapted Xcc and non-adapted Xcv strains, and cellular changes were more prominent in the leaves inoculated by the non-adapted Xcv Bv5-4a.1 (Fig. 2).

Fig. 2

Preferentially elevated oxidative stress in the Chinese cabbage leaves during the non-adapted bacteria-triggered hypersensitive cell death. (A) Relative electrolyte leakages (%) and (B) lipid peroxidation in the leaves inoculated with an adapted strain X. campestris pv. campestris (Xcc) 8004, and a non-adapted strain X. campestris pv. vesicatoria (Xcv) Bv5-4a.1. Chinese cabbage leaves were syringe-infiltrated with bacterial suspension (10⁷ cfu/ml) of Xcc and Xcv and harvested at 1, 6 and 24 h. Sterile water was infiltrated as a mock inoculation. TBARS, thiobarbituric acid reactive substances. The data are the means ± standard errors of the four independent experimental replicates. Each experiment has four replications. Means with the same letter above the bars are not significantly different at the 5% level by the least significant difference tests.

Electrolyte leakages from the inoculated leaves to investigate changes in membrane stability (Fig. 2A). Electrolyte leakages did not occur in the leaves inoculated by Xcc or Xcv at 1 h compared to the mock-inoculated leaves, and a slight reduction was detected in the mock-inoculated leaves at 6 h. Electrolyte leakages from the mock-inoculated leaves at 24 h, returned to the level at 1 h. Increased electrolyte leakages were found in the Xcc-inoculated leaves at 24 h compared to those from mock-inoculated leaves, and Xcv inoculation drastically increased electrolyte leakages compared to those from mock- and Xcc-inoculated leaves.

Lipid peroxidation as a biochemical marker for oxidative stress was compared in the inoculated leaves by measuring amounts of TBARS (Fig. 2B). Lipid peroxidation did not occur in the leaves by Xcc or Xcv until 6 h compared to the mock-inoculated leaves. At 24 h, Xcc inoculation slightly increased lipid peroxidation at 24 h compared to the mock inoculation, and Xcv markedly elevated lipid peroxidation to a significantly higher level compared to the Xcc inoculation.

Different transcriptional regulation of defense-related genes in Chinese cabbage leaves by adapted Xcc and non-adapted Xcv

Expression of six Chinese cabbage defense-related genes, BrPR1, BrPR4, BrChi1, BrGST1, BrAPX1 and BrVSP2, was analyzed in the leaves at 24 h after mock, Xcc and Xcc inoculation (Fig. 3). Five genes BrPR1, BrPR4, BrChi1, BrGST1 and BrAPX1 showed similar expression patterns. Moderate inductions by Xcc and drastic increases by Xcv were commonly found compared to mock inoculation. By contrast, BrVSP2 gene expression was significantly reduced by Xcc compared to mock inoculation, and much less gene expression was followed by Xcv.

Fig. 3

Expression of defense-related genes in the Chinese cabbage leaves inoculated by the adapted and non-adapted strains of Xanthomonas campestris pathovars. A virulent adapted strain 8004 of X. campestris pv. campestris (Xcc) and a non-adapted strain Bv5-4a.1 of X. campestris pv. vesicatoria (Xcv) were syringe-infiltrated into the abaxial surface of the primary leaves of Chinese cabbage seedlings. Gene expressions were analyzed using leaf tissues harvested at 24 h. BrPR1, Brassica rapa pathogenesis-related protein 1 gene; BrPR4, Brassica rapa pathogenesis-related protein 4 gene; BrChi1, Brassica rapa chitinase 1 gene; BrGST1, Brassica rapa glutathione S-transferase 1 gene; BrAPX, Brassica rapa ascorbate peroxidase gene; BrVSP2; Brassica rapa vegetative storage protein 2 gene. The data are the means ± standard deviations of the three independent experimental replicates. Means with the same letter above the bars are not significantly different at the 5% level by the least significant difference tests.

Day length-regulated HCD and bacterial growth in the leaves inoculated by non-adapted Xcv

The effect of light length on the Xcv-triggered HCD and non-adapted bacterial growth in the Chinese cabbage leaves was evaluated (Fig. 4). Prolonged light and dark environments differently changed the HCD in the Xcv-inoculated leaves compared to the standard light length (12-h day/12-h night) (Fig. 4A). Continuous light did not alter the electrolyte leakages in the mock-inoculated leaves at 8 h, but continuous darkness slightly increased the electrolyte leakages compared to the standard light length. The distinct increases in the electrolyte leakages in the Xcv-inoculated leaves under the standard light length compared to mock-inoculation were detected at 8 h. However, the Xcv-mediated elevated electrolyte leakages were not changed by continuous light and dark conditions at 8 h. At 24 h, continuous light slightly attenuated the electrolyte leakages from the mock-inoculated leaves, but constant darkness increased the electrolyte leakages compared to the standard light length. Remarkable increases in the electrolyte leakages appeared in the Xcv-inoculated leaves under the standard light length at 24 h compared to 8 h. The continuous light decreased the electrolyte leakages from the Xcv-inoculated leaves compared to the standard light length, but continuous dark elevated the electrolyte leakages. Altered light length also changed the in planta bacterial growth of Xcv in the Chinese cabbage leaves (Fig. 4B). Bacterial growth of Xcv was reduced in the leaves at 24 h after inoculation compared to 0 h under the standard light length. The continuous light environment could not change the in planta Xcv bacterial population compared to the standard light length. In contrast, the Xcv bacterial growth was notably accelerated under constant dark conditions.

Fig. 4

Non-adapted bacteria-triggered hypersensitive cell death and in planta bacterial growth in Chinese cabbage leaves regulated by ambient light environment. (A) Relative electrolyte leakages (%) in the leaves inoculated by a non-adapted strain Bv5-4a.1 of Xanthomonas campestris pv. vesicatoria (Xcv) at 8 and 24 h under different light period regimes. (B) Non-adapted bacterial growth in the inoculated leaves under different light period regimes. The data are the means ± standard errors of the four independent experimental replicates. Means with the same letter above the bars are not significantly different at the 5% level by the least significant difference tests.

RH-regulated HCD and bacterial growth in the leaves inoculated by non-adapted Xcv

The effect of RH on the Xcv-triggered HCD and non-adapted bacterial growth in the Chinese cabbage leaves was evaluated (Fig. 5). The HCD occurrence and tissue browning at the Xcv-inoculated areas were distinctly attenuated by prolonged high RH (Fig. 5A). Under moderate RH conditions, visible leaf damage (red arrows) was found only in the Xcv-inoculated leaves at 24 hpi, and brown discoloration (green arrows) in the leaves with the tissue collapse was evident after the ethanol clearing. The visible leaf damage (red arrows) and brown discoloration (green arrows) in the Xcv-inoculated leaves were also observed at 24 hpi under high RH conditions for 8 h. Water-soaked symptoms (white arrows) were found in the mock- and Xcv-inoculated leaves at 8 hpi under high RH conditions for 8 h. No visible leaf damage and brown discoloration were observed in the mock- and Xcv-inoculated leaves at 24 hpi under high RH conditions for 24 h, and only water-soaked symptoms (white arrows) were found. The occurrence of HCD in the Xcv-inoculated leaves was delayed by the continued high RH, which was shown by decreased electrolyte leakages from the inoculated leaves (Fig. 5B). Under a high RH for 8 h, changes in electrolyte leakages were not detected in the mock-inoculated leaves at 8 h compared to the moderate RH condition. Without the high RH, electrolyte leakages were slightly increased in the mock-inoculated leaves at 24 h compared to 8 h. The high RH for 8 h did not affect the electrolyte leakages in the mock-inoculated leaves at 24 h. However, continuous high RH for 24 h slightly reduced electrolyte leakages in the mock-inoculated leaves. Xcv inoculation increased the electrolyte leakages at 8 h without a high RH. However, the high RH for 8 h suppressed the Xcv-triggered electrolyte leakages. The electrolyte leakages were significantly elevated in the Xcv-inoculated leaves at 24 h without a high RH compared to 8 h. The high RH for 8 h could not alter the Xcv-triggered electrolyte leakages compared to the usual RH condition, whereas continuous high RH for 24 h considerably decreased the electrolyte leakages. The increasing high RH period compromised the bacterial growth of non-adapted Xcv limited in the Chinese cabbage leaves (Fig. 5C). In the absence of high RH, the Xcv bacterial population was maintained in the leaves at 24 h. By contrast, the high RH for 8 h moderately raised the Xcv bacterial growth compared to the moderate RH condition. The Xcv bacterial number was significantly augmented in the leaves under continuous high RH for 24 h compared to the 8 h high RH.

Fig. 5

Non-adapted bacteria-triggered hypersensitive cell death (HCD) and in planta bacterial growth in Chinese cabbage leaves regulated by relative humidity (RH) environment. (A) Altered HCD and tissue discoloration in the leaves inoculated by a non-adapted strain Bv5-4a.1 of Xanthomonas campestris pv. vesicatoria (Xcv) under different RH regimes. White arrows, water-soaked areas; red arrows, HCD-occurred areas; green arrows, brownish discolored areas. (B) Relative electrolyte leakages (%) in the Xcv-inoculated leaves at 8 and 24 h under different RH regimes. (C) Non-adapted bacterial growth in the inoculated leaves under different RH regimes. The data are the means ± standard errors of the four independent experimental replicates. Means with the same letter above the bars are not significantly different at the 5% level by the least significant difference tests.

Effect of fluridone and ABA pretreatment on the HCD and bacterial growth in the leaves inoculated by a non-adapted Xcv

ABA is known to be involved in the light and RH responses of plants. To investigate the involvement of ABA in the establishment of HCD and non-adapted Xcv bacterial growth in the leaves, fluridone (ABA inhibitor) and ABA were exogenously applied to the seedlings at 1 day before Xcv inoculation. The electrolyte leakages from the Xcv-inoculated leaves treated with mock, fluridone and ABA were measured at 8 and 24 h and the bacterial proliferation was evaluated at 24 h (Fig. 6).

Fig. 6

Abscisic acid (ABA)-regulated non-adapted bacteria-triggered hypersensitive cell death and in planta bacterial growth in Chinese cabbage leaves. (A) Relative electrolyte leakages (%) in the leaves inoculated by a non-adapted strain Bv5-4a.1 of Xanthomonas campestris pv. vesicatoria (Xcv) at 8 and 24 h after 1 day pretreatment with different concentrations (0, 5, 10, 20, 50 and 100 μM) of fluridone and ABA. (B) Non-adapted bacterial growth in the inoculated leaves pretreated with fluridone and ABA. The data are the means ± standard errors of the five independent experimental replicates. Means with the same letter above the bars are not significantly different at the 5% level by the least significant difference tests.

Fluridone pretreatment increased the Xcv-triggered electrolyte leakages in the leaves, but ABA pretreatment alleviated the electrolyte leakages at 8 and 24 h (Fig. 6A). At least 10 μM of fluridone increased the electrolyte leakages from the Xcv-inoculated leaves compared to the mock (0 μM) at 8 h. Although increasing fluridone concentrations to 20–100 μM, the elevated level of electrolyte leakages by 10 μM of fluridone was maintained. Fluridone pretreatment with 5 to 20 μM did not change the electrolyte leakages compared to the mock (0 μM), but higher fluridone doses (50 and 100 μM) augmented the electrolyte leakages from the Xcv-inoculated leaves at 24 h. Pretreatment with ABA (10 to 100 μM) gradually reduced the electrolyte leakages in a dose-dependent manner at 8 h. Only the highest ABA concentration (100 μM) could attenuate the electrolyte leakages slightly compared to the mock (0 μM) at 24 h.

Non-adapted Xcv bacterial growth in the leaves was not altered by fluridone pretreatment but reduced by ABA pretreatment (Fig. 6B). At 24 h, Xcv bacterial growth was not changed in the leaves pretreated with two doses (10 and 100 μM) of fluridone compared to the mock treatment (0 μM). The Xcv bacterial population in the leaves pretreated with 10 μM of ABA was not changed, and the higher ABA to 100 μM slightly reduced Xcv growth (0.92 log-fold) in the leaves at 24 h.

Discussion

Chinese cabbage plants, an economically important vegetable, have suffered from different diseases, including black rot caused by the bacteria Xanthomonas campestris pv. campestris (Xcc). Xanthomonas campestris pv. vesicatoria (Xcv) causes bacterial spots in susceptible cultivars of tomato and pepper plants but triggers HCD in non-host plants to enhance plant immunity. A high Xcv titer (more than 10⁷ cfu/ml) induced HCD in the non-host bean leaves (Whalen et al., 1988). A high titer (4 × 10⁸ cfu/ml) of Xcv infiltrated into the non-host Nicotiana benthamiana leaves led to HCD, but the Xcv mutant deficient in effector protein XopX could not induce the HCD (Metz et al., 2005). Non-host N. tabacum leaves also showed HCD by infiltrating a high Xcv titer (10⁸ cfu/ml), and the generation of reactive oxygen species was suggested as an HCD-inducing factor in response to the non-adapted Xcv (Zurbriggen et al., 2009). More than 10⁶ cfu/ml of Xcv elevated electrolyte leakage at the non-host Citrus sinensis leaves, indicating HCD occurrence (Daurelio et al., 2013). In our previous study, two strains 85-10 and Bv5-4a.1 of Xcv were inoculated in the Chinese cabbage leaves by two different inoculation methods, and Xcv-triggered plant responses were compared with Xcc-inoculated leaves under disease development (Lee and Hong, 2012). A virulent Xcc strain 8004 mediated progressive chlorotic symptoms at the inoculated Chinese cabbage leaves by leaf clipping method, whereas none of the visual change was found in the leaves clipping inoculated with the two Xcv strains. It seems that the non-adapted Xcv bacterial invasion was halted at the main vein of the Chinese cabbage leaves by the early activated defense. By contrast, syringe-infiltrating a high Xcv titer into the leaves resulted in the occurrence of HCD, but no apparent change was observed in the Xcc-inoculated leaves at the same time point. In the current study, high titers of Xcc and Xcv were infiltrated into the Chinese cabbage leaves to investigate different plant responses against the adapted and the non-adapted bacteria via the direct contact of the bacterial cells with non-host plant cells. Xcv-triggered HCD was observed within 24 h, as we found in our previous study. Generally, a low titer of non-adapted bacteria, such as 10⁴ and 10⁵ cfu/ml, was infiltrated into leaves of various non-host plants to evaluate whether growth of non-adapted bacterial species are indeed suppressed in the inoculated leaves so far, and it successfully has shown the limited or delayed growth of the non-adapted bacteria (Gupta et al., 2013; Lee et al., 2022a; Whalen et al., 1988). However, it is difficult to find that HCD was observed macroscopically in the non-host plant leaves inoculated by the low titer of the non-adapted bacteria. In our previous study, in planta bacterial growth of Xcc and Xcv were compared in the leaves by infiltrating bacterial suspensions (10⁵ cfu/ml), and a distinct difference between Xcc proliferation and Xcv suppression was shown over time. No distinct HCD was found in the Chinese cabbage leaves inoculated by Xcv by 14 dpi. In the current study, a high titer (10⁷ cfu/ml) of the bacterial suspensions was applied to evaluate bacterial growth of Xcv in the HCD-appearing leaves, and compared with the adapted Xcc bacterial growth in the inoculated leaves. A slight reduction of Xcc growth at 6 h may be due to activated basal immunity at the early hour. The slight and temporary suppression of Xcc strain 8004 growth was also found in the susceptible Arabidopsis ecotype Col-0 at early inoculation time (Lummerzheim et al., 1993). Several pattern-triggered immunity, mediated by the pattern and corresponding receptor interactions, were revealed in Arabidopsis, and the receptor homologs against Xcc were suggested in the Chinese cabbage genome (Kim et al., 2020). The basal plant immunity transiently activated by phytopathogenic bacteria, including Xcc, needs further elucidation in Chinese cabbage plants. Xcc could overcome the basal immunity at 24 h, showing increased growth, followed by progressive symptom appearance a few days later. Surprisingly, Xcv growth increased in the leaves transiently at 6 h when HCD did not occur, although the Xcv growth declined soon with HCD occurrence in the inoculated leaves at 24 h. Non-adapted bacteria Xanthomonas oryzae pv. oryzae (Xoo) in the C. sinensis leaves drastically decreased after inoculation (Rani and Podile, 2014). However, Chinese cabbage leaves are likely to allow a proliferation of the non-adapted Xcv temporarily; they have time to prepare and trigger HCD to suppress the bacterial growth of Xcv actively. It is similar to the slightly increased and maintained growth of a non-adapted bacteria Xcc within the non-host leaves of the C. limon (Chiesa et al., 2019).

Many mesophyll cells were dying by Xcv at 24 h, and it was comparatively higher than dying cell numbers in the Xcc-inoculated leaves. Notably, HCD with tissue collapse was found in the Xcv-inoculated leaves, but not all mesophyll cells were stained. Only massively collapsed cells stained in the Xcv-inoculated leaves were observed, but many degenerating cells were not stained. The non-adapted Xcv may still be alive in the degenerating cells as evidenced by the steady status of Xcv bacterial growth level at 24 h. It may need further evaluation of the Xcv bacterial growth after 24 h to see distinct and complete suppression of the non-adapted Xcv in the Chinese cabbage leaves established with HCD.

Increased electrolyte leakage and lipid peroxidation have been physiological hallmarks of plant HCD in response to invasion of avirulent or non-adapted pathogen strains. Rapidly increased electrolyte leakage indicating loss of cell membrane integrity was seen during the non-adapted pathogen-related HCD establishment (Chiesa et al., 2019; Gupta et al., 2013; Krzymowska et al., 2007). Lipid peroxidation associated with the HCD has been described with or without increased electrolyte leakage. The augmented electrolyte leakage and lipid peroxidation were concomitantly found in the cucumber cotyledons by a non-adapted Pseudomonas syringae pv. pisi, but these reactions were not prominent by a virulent strain of P. syringae pv. lachrymans (Keppler and Novacky, 1986). HCD appeared in the Citrus sinensis leaves in response to a non-adapted bacteria Xoo and significant increase in lipid peroxidation was found in the Xoo-inoculated C. sinensis leaves at the early hours after the bacterial inoculation (Rani and Podile, 2014; Rani et al., 2015). But lipid peroxidation by an adapted X. axonopodis pv. citri (Xac) was only slightly increased at the later infection stage (Rani et al., 2015). Highly increased lipid peroxidation in the Xcv-inoculated Chinese cabbbage leaves suggests involvement of oxidative burst in the HCD-associated events. Increased production of H₂O₂ was detected in the Arabidopsis leaves inoculated by non-adapted bacteria P. syringae pv. tabaci and P. syringae pv. glycinea, but no H₂O₂ production was compared in the leaves inoculated by a virulent adapted strain of P. syringae (Ishiga et al., 2016). A preferential H₂O₂ increase was found in the Xoo-inoculated C. sinensis leaves, in line with lipid peroxidation during the non-host plant immunity (Rani et al., 2015). Two antioxidant enzymes, peroxidase and superoxide dismutase, were highly activated in the non-adapted Xoo-inoculated C. sinensis leaves compared to those in the adapted Xac-inoculated leaves (Rani et al., 2015). Significantly up-regulated BrGST1 and BrAPX1 gene expression in the Xcv-inoculated leaves shows indirect evidence of the relatedness of antioxidant responses in the non-host plant immunity of Chinese cabbage. Mung bean Cu/Zn-SOD and APX genes were drastically up-regulated in the leaves by non-adapted X. hortorum pv. pelargoni as compared to those by adapted X. axonopodis pv. phaseoli, suggesting the roles of activated antioxidant responses in the non-host plant immunity (Farahani and Taghavi, 2016). Investigation of the accumulation of reactive oxygen species and antioxidant responses in the Xcv-inoculated leaves will provide insight into the biochemical immunity of Chinese cabbage plants.

Drastically increased expression of defense-related genes BrPR1, BrPR4 and BrChi1, was also one of the molecular strategies in Chinese cabbage leaves against the non-adapted Xcv. PR1 was also highly up-regulated in the C. limon leaves inoculated with a non-adapted X. campestris pv. campestris, but a virulent adapted strain of X. citri induced relatively low PR1 gene expression (Chiesa et al., 2019). PR1a, PR1b and PR2b genes in tomato leaves were highly expressed in response to a non-adapted P. syringae pv. tabaci inoculation, but their induction by a virulent adapted strain Pst was relatively weak (Lee et al., 2013). These differentially up-regulated PR genes were known to encode antimicrobial peptides trafficking to different cellular compartments (Ali et al., 2018; Han et al., 2023; Kim and Hwang, 2015; Pečenková et al., 2022). PR proteins can form complexes with other PR protein or non-PR protein members to enhance plant immune responses (Hwang et al., 2014; Ma et al., 2018; Wang et al., 2020). These findings suggest that simultaneously up-regulated distinct PR gene expressions might have cooperated with each other to efficiently halt invading non-adapted pathogens via antimicrobial activities and/or plant cell death regulation. PR1a and PR4 genes increased strongly in Chinese cabbage leaves inoculated with a non-adapted Pst, and PR1a, PR1b, PR5a and PR5b gene expressions were induced in tobacco leaves by a non-adapted Psm (Krzymowska et al., 2007; Park et al., 2005). However, these PR gene expression during the non-host plant immunity was not compared to expressions in the Chinese cabbage and tobacco leaves during the susceptible responses. Preferential transcriptional activation of these PR genes in the present study might efficiently establish non-host plant immunity against the non-adapted Xcv in Chinese cabbage leaves. Several genes encoding functionally unknown proteins were identified in the Pst-inoculated Chinese cabbage leaves during the HCD-mediated non-host immunity (Park et al., 2005). Novel genes involved in non-host immunity were discovered from Xac-inoculated tobacco and Xcv-inoculated C. sinensis leaves (Daurelio et al., 2011, 2013). Transcriptome analysis of Chinese cabbage genes regulated explicitly by the Xcv-triggered HCD will provide a clue on the molecular basis of non-host immunity of Chinese cabbage plants against the non-adapted bacterial species. In contrast to the other PR genes, drastically reduced BrVSP2 expression in the Xcc- and Xcv-inoculated Chinese cabbage leaves may suggest suppressed jasmonic acid signaling and/or activated salicylic acid signaling during the basal and non-host plant immunity (Cao et al., 2016; Leon-Reyes et al., 2010). Notably, Arabidopsis AtVSP2 and Chinese cabbage BrVSP2 were inducible genes by attacks from various insect herbivores with chewing and piercing/sucking behavior (Abe et al., 2008, 2009; Cao et al., 2016; Verhage et al., 2011). Investigating the responses of Chinese cabbage plants to the invasion by various pathogens or insects into the Xcc- or Xcv-inoculated leaves will provide insight into multiple plant interactions with microbes and insects in Chinese cabbage fields as suggested in a variety of plant-pathogen-insect interaction systems (Desurmont et al., 2016; Franco et al., 2017; Ontiveros et al., 2022; Sun et al., 2016).

Xcv-secreted effectors and Chinese cabbage interacting partners that trigger the HCD accompanying lipid peroxidation and PR gene activation have still not been uncovered. The effectors XopB, XopJ and XopS delivered by Xcv attenuated immunity in the susceptible pepper plant to facilitate bacterial invasion and in planta proliferation (Kay and Bonas, 2009; Priller et al., 2016; Raffeiner et al., 2022; Schulze et al., 2012; Üstün et al., 2013). Searching for Xcv effectors triggering HCD and non-host immunity in Chinese cabbage plants should be investigated, as demonstrated in recognizing Xcv effectors avrRxv and XopQ by non-host plants (Adlung et al., 2016; Whalen et al., 1988).

Environmental conditions have influenced plant immunity against pathogen attacks. In particular, changes in light conditions, such as different light intensities and periods, altered disease susceptibility and resistance of host plants. Low light intensity increased tomato bacterial specks showing more severe lesions and in planta bacterial growth compared to standard light (Luo et al., 2023). Continuous light ameliorated bacterial specks in Arabidopsis leaves inoculated by a virulent Pst strain, whereas continuous dark accelerated symptom development compared to the usual light regime (Lajeunesse et al., 2023). By contrast, in planta Pst bacterial population in Arabidopsis leaves was reduced by the continuous light, but was not affected by constant dark (Lajeunesse et al., 2023). Continuous dark increased bacterial growth of X. campestris pv. malvacearum (Xcm) in the resistant cotton cultivar compared to the usual light/dark regime, but the continuous dark did not change highly proliferated bacteria in the susceptible cultivar (Morgham et al., 1988). However, there was no information on whether the compromised Xcm bacterial growth in the resistant cultivar under continuous dark is associated with altered HCD. Immediate bacterial inoculation after illumination initiation led to higher salicylic acid accumulation and earlier PR-1 gene expression in Arabidopsis inoculated with an avirulent Psm than the inoculation at later times, which was closely associated with HCD occurrence and the suppressed in planta bacterial growth under a prolonged light period (Griebel and Zeier, 2008). Transiently expressed Zymoseptoria tritici effector protein candidates in non-host N. benthamiana leaves triggered HCD under 16 h-day light condition, but continuous dark suppressed the HCD (Kettles et al., 2017). These indicated that a limited light period and intensity might provide favorable environments to virulent bacteria during the pathogenesis, but prolonged light periods and high light intensity seem to contribute to plant immunity to an avirulent strain triggering HCD in their hosts. HCD associated with plant immunity was also regulated by light conditions. Nevertheless, the involvement of ambient light conditions in the HCD-mediated NHR is rarely described so far. Unexpectedly, the light period is likely to negatively regulate HCD in Chinese cabbage leaves against Xcv, in the present study. Reduced HCD under the prolonged light period and accelerated HCD under complete darkness were found in the Xcv-inoculated Chinese cabbage leaves. It was contrary to the long day-dependent HCD in the resistant host plants against avirulent strains. Notably, continuous light and dark also reduced and increased the electrolyte leakages from the mock-inoculated leaves at 24 h, respectively, indicating that the light period alone can regulate plant membrane permeability regardless of the non-adapted Xcv inoculation in the Chinese cabbage leaves. It remains how light modulates the membrane permeability of Chinese cabbage leaves with or without Xcv inoculation. In planta bacterial population of the non-adapted Xcv drastically increased in the Chinese cabbage leaves under continuous dark, although continuous light could not affect the Xcv bacterial proliferation. Illumination for 12 h was sufficient to establish NHR in Chinese cabbage leaves against non-adapted Xcv. We did not determine the minimum dark period for the compromised Xcv bacterial growth suppression. Still, a critical continuous dark period necessary to lower NHR will be more than 12 h. In recent decades, increasing evidence has suggested a connection between the light environment and plant immunity associated with molecular recognition and signaling (Ahammed et al., 2018; Gao et al., 2020; Iqbal et al., 2021). Unidentified key determinants involved in the NHR against Xcv might be down-regulated or degraded under more extended dark periods of more than 12 h, and searching these molecules will provide more insights into the deployment of NHR.

High RH has profoundly enhanced plant disease susceptibility. Rice blast, tomato grey mould and Arabidopsis bacterial speck on leaves developed progressively under the higher RH (Li et al., 2023; Qiu et al., 2022; Yao et al., 2023). Prolonged leaf wetness provided favorable environments for Asiatic citrus canker by X. smithii ssp. citri and peach-almond hybrid bacterial spot by X. arboricola pv. pruni (Dalla Pria et al., 2006; Morales et al., 2018). RH-modulated HCD triggered in plants by avirulent adapted strains was rarely described. Intercellular washing fluids from the susceptible tomato leaves inoculated by Cladosporium fulvum race 0 caused grey necrosis in the Cf-9 gene-harbouring tomato leaves under 70% RH, whilst only slight chlorosis was developed on the leaves of the same tomato genotype under 98% RH, suggesting Cf-9/Avr9 interaction-dependent HCD delayed and reduced by high RH (Hammond-Kosack et al., 1996). Tomato seedlings simultaneously overexpressing tomato Cf-9 gene and C. fulvum Avr9 gene displayed necrotic spots on the cotyledons at 2–3 days after emergence under 70% RH, but increased RH to 95% delayed the HR to 14–16 days after emergence (Wang et al., 2005). Arabidopsis lesion mimic mutant cpn1-1 accelerated HCD against an avirulent Psm carrying avrRpt2 compared to wild-type, but the accelerated HCD in the cpn1-1 was compromised under high RH (Jambunathan et al., 2001). Another Arabidopsis lesion mimic mutant ssi4 also showed spontaneous cell death in the absence of pathogen attack under moderate RH, but the cell death did not appear under high RH (Zhou et al., 2004). RH-dependent HCD regulation against invading non-adapted pathogens has not been described in the plants so far. In the present study, increased wetness negatively influenced the HCD as well as plant immunity in Chinese cabbage leaves against the non-adapted Xcv. It matched previous findings with high RH-mitigated HCD and/or plant immunity in plants. The high RH may suppress the activation of NHR-associated responses, such as defense-related gene expression and defense signaling molecule level (Jambunathan et al., 2001; Qiu et al., 2022; Zhou et al., 2004). It will be worth investigating whether lipid peroxidation and defense-related gene expression highly up-regulated during the Xcv-triggered HCD in the Chinese cabbage leaves were also dependent on light and RH conditions.

During the HCD establishment by the non-adapted Xcv, ABA production and stomatal closure may be differently regulated under different light/dark regimes in the Chinese cabbage leaves. ABA levels were elevated by extended darkness in several plants (Weatherwax et al., 1996; Yang et al., 2000). The continuous dark can accelerate stomatal closure by activating ABA signaling, which leads to lessening water loss caused by the HCD occurrence in the leaves. Surprisingly, the continuous dark for 24 h accelerated HCD in the Xcv-inoculated leaves. Highly proliferated Xcv within the Chinese cabbage leaf tissues under constant dark conditions can derive accelerated HCD.

High RH has activated oxidative catabolism of ABA via 8′-hydroxylase genes and reduced ABA levels in Arabidopsis leaves (Arve et al., 2015; Okamoto et al., 2009). Lowered ABA by ambient high RH perturbed usual plant behavior, including the stomatal response in guard cells (Okamoto et al., 2009). High RH-suppressed pre-invasive plant immunity was correlated with compromised ABA-mediated stomatal closure in response to bacterial invasion from the leaf surface. Still, the high RH could not change the apoplastic population of the bacteria inoculated by leaf infiltration (Panchal et al., 2016). In the present study, the non-adapted Xcv was syringe-infiltrated into the leaves for direct contact of Xcv with plant mesophyll cells, and it could lead to HCD and suppressed bacterial growth. To investigate the association of ABA with high RH-suppressed HCD and plant immunity against Xcv in the Chinese cabbage leaves, ABA and fluridone were exogenously applied before Xcv inoculation under moderate RH conditions. It demonstrates that ABA attenuates HCD in the Chinese cabbage leaf cells encountered with the non-adapted Xcv. The endogenous and pretreated ABA may play in the guard cells and contribute to stomatal closure, and make keep the internal moisture of the Xcv-inoculated leaves intact and protect the leaves from HCD accompanied by tissue collapse. It was the opposite effect of high RH on the Xcv-triggered HCD. Although the high RH inhibits ABA accumulation in the leaves and makes stomata open, the high RH alone around the Xcv-inoculated leaves could preserve the leaves from water loss due to HCD occurrence and delay the HCD. However, ABA may induce stomatal closure to minimize water loss caused by the HCD under moderate RH conditions. Three ABA 8′-hydroxylase genes differently regulated by heat stress were found in two Chinese cabbage lines (Dong et al., 2015). More insights on the altered Xcv-triggered HCD by high RH and ABA can be provided by studies on the regulation of the Chinese cabbage ABA 8′-hydroxylase under different RH regimes and changed endogenous ABA levels. More recently, it has been found that Pst-secreted effector AvrPtoB targets ABA 8′-hydroxylase to promote the bacterial virulence in Arabidopsis (Liu et al., 2022). Investigating Xcv effectors regulating Chinese cabbage HCD and plant immunity through ABA signaling pathways will also pave the way to decipher the role of Xcv bacterial effectors in NHR differently regulated by environmental conditions.

ABA has negatively affected basal plant immunity against bacterial infection in plant mesophyll cells by inhibiting salicylic acid defense signaling as a post-invasive defense (Cao et al., 2011). Generally, the disease susceptibility of host plants against Xanthomonas spp. was enhanced by ABA. Citrus canker development by X. citri subsp. citri was promoted in the ABA-treated orange leaves, concomitant with increased jasmonic acid and decreased salicylic acid (Long et al., 2019). Exogenous ABA increased lesion length of rice leaf blight by Xoo, whereas reduced endogenous ABA by fluridone induced disease resistance (Xu et al., 2013). By contrast, neither ABA nor fluridone changed the rice leaf blight severity in resistant rice plants (Xu et al., 2013). It has rarely been described that ABA acts as a positive regulator of plant immunity against pathogens, including non-adapted pathogens, except for the role in stomatal closure. ABA-induced resistance was found in tomato plants to Alternaria solani and Arabidopsis to Meloidogyne paranaensis (de Souza Yop et al., 2023; Song et al., 2011). In this study, exogenous ABA enhanced plant immunity against the non-adapted Xcv in mesophyll cells of Chinese cabbage leaves, showing the reduced Xcv population. Xcv-triggered HCD attenuated by exogenous ABA indicates that ABA plays different roles in HCD occurrence and in planta bacterial suppression in Chinese cabbage leaves against the non-adapted Xcv. The increased Xcv bacterial growth in the leaves under the prolonged high RH may derive from reduced ABA levels in the mesophyll cells. Increased activities of phenylalanine ammonia-lyase, peroxidase and polyphenol oxidase were suggested as tomato defence responses during the ABA-induced resistance to the early blight (Song et al., 2011). G-protein α-subunit is required for the ABA-induced resistance in rice plants against brown spots (de Vleesschauwer et al., 2010). How exogenous ABA could enhance non-host plant immunity in the Chinese cabbage mesophyll cells remains elucidated.

In conclusion, Chinese cabbage seedlings responded differently to the adapted and non-adapted pathovars of Xanthomonas campestris, showing different physiological and molecular features (Fig. 7). No drastic change in plant cell death, oxidative stress and defence gene expression were demonstrated in the adapted Xcc-inoculated leaves, but the in planta bacterial growth was allowed without visible symptoms at 24 h. By contrast, plant immunity against the non-adapted Xcv distinctly occurred, accompanied by HCD, lipid peroxidation, and highly activated defense gene expressions in the leaves. The HCD occurrence and suppressed non-adapted bacterial proliferation in the leaves were modulated by environmental conditions such as light length and RH and by endogenous colonization of the adapted Xanthomonas species. ABA is likely to be involved in the HCD and non-adapted Xcv bacterial growth in Chinese cabbage leaves. Further investigation on different physiological, metabolic and transcriptional changes of Chinese cabbage leaves in response to the adapted Xcc and non-adapted Xcv will provide a better understanding of the onset of HCD and defense signaling in NHR.

Fig. 7

Proposed model for different responses of Chinese cabbage leaves to an adapted strain 8004 of Xanthomonas campestris pv. campestris (Xcc) and a non-adapted strain Bv5-4a.1 of X. campestris pv. vesicatoria (Xcv). Syringe-inoculation with a high titer (10⁷ cfu/ml) of Xcc 8004 led to bacterial proliferation within the leaf tissues at 24 h, when plant cells were not disintegrated. In contrast, the same titer (10⁷ cfu/ml) of Xcv Bv5-4a.1 triggered hypersensitive cell death (HCD) in the leaf tissues, which can be altered by ambient environments such as light and wetness conditions as well as endogenous abscisic acid (ABA). Significantly elevated electrolytes, lipid peroxidation and defense gene expression, were found in the HCD-occurred leaves. The non-adapted bacterial cell proliferation was suppressed in the Chinese cabbage leaves during the establishment of HCD, which was comparable with Xcc growth. RH, relative humidity.

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