Xcc infection can occur at any developmental stages of the plant, starting with germination of the infected seeds to maturity. Xcc primarily spreads from the infected seeds, which is a major route of disease transmission. However, black rot can also be transmitted through infested soil, crop residues, and by various environmental and mechanical means via wind, insects, aerosols, irrigation water, rain, and farm equipments (Vicente and Holub, 2013). The pathogen can survive longer in plant debris in soil than as free-living cells for up to 2 years. The germination of colonised seeds leads to the infection of the seedlings. The bacteria mostly enter the plants through the hydathodes on the leaf margins although it can also invade the plant through the wounds caused by machinery, insects, animals, rain, irrigation, and wind, etc. Through these entry points, the bacteria spread intercellularly, colonize the mesophyll first, and then gain access to the plant vascular systems and multiply in the vessels leading to the rapid spreading of systemic host infection. At the same time, the xylem disintegrates, spreading the bacteria between the surrounding parenchyma cells, killing the cells, and causing cavities to be formed (Agrios, 2005). The symptoms are manifested by very distinctive appearance, i.e., V-shaped chlorotic to necrotic yellow lesions originating from the leaf margins and progressing toward the middle vein of the leaves. Also, the symptoms include the darkening of veins of the leaves and of the vascular tissue of the stem. Due to necrosis, the leaves fall prematurely, and systemic infection may lead to stunted growth of the plants. During warm and humid climates, Xcc thrives as a severe disease agent and the bacteria often ooze out to the surface of the leaves through the hydathodes/wounds and subsequently spread in droplets of guttation to the neighbouring plants by wind, rain, or water splashes. Black rot affecting different varieties of B. oleracea with the typical V-shaped lesion is depicted in Figures 2A–D.

Several pathogenic races of Xcc were reported in different Brassica species and Kamoun et al. (1992) first proposed the race structure of Xcc. Initially, six races (1–6) were identified by Vicente et al. (2001). Later, three more races (7–9) were identified by Fargier and Manceau (2007) and recently, two novel races, race 10 and 11 were reported in Portugal (Cruz et al., 2017). Presently, eleven different physiological races infecting Brassica species have been reported (Vicente et al., 2001; Fargier and Manceau, 2007; Cruz et al., 2017), indicating the complexity of Xcc. Among all, races 1 and 4 are the most aggressive and predominant worldwide (Lema et al., 2012a; Vicente and Holub, 2013) though their frequencies in B. oleracea varies with the geographical region.

Genomics pertains to the study of all the genes in a genome, including the identification of gene sequences, gene structures, and annotations. It plays an important role in discovering the genetic variation underlying important traits and contribute to the genetic improvement of crop species. Rapid progress in the NGS technologies has expanded our ability to understand the whole genome and helps in bridging the gap between the genotype and phenotype. Genomic revolution has led to the generation of whole-genome sequences, expressed sequence tags (ESTs), large-insert genomic libraries, high density genetic maps, and millions of molecular markers which could be used for bi-parental/association mapping, cloning of genes/QTLs, and genomic selection, etc. for agronomically important traits in different crops. The identification of QTLs, allelic variation in the genes governing the trait of interest will enhance the possibilities of improvement of Brassica species especially for disease resistance.

Transcriptomics refers to the study of the entire set of RNA of an organism, including mRNAs and other non-coding RNAs (McGettigan, 2013). Transcriptome study helps in characterizing and quantifying the entire RNA present in an organ, tissue, or cell in a given organism. Different genes of a cell are up or downregulated in different physiological and developmental process. So, comparing of RNA expression profile provides an insight into when and where the genes are expressed under different treatments and helps in validation of the putatively differentially expressed genes. This, in turn, helps in the identification of candidate genes influencing any important traits involved in the cellular process of an organism. Transcriptome is highly dynamic unlike the genome which basically remains constant irrespective of age, organ, or growth conditions (El-Metwally et al., 2014). RNA-seq has largely replaced the earlier commonly used approaches for gene expression profiling such as microarray and serial analysis of gene expression (SAGE). Transcriptome analysis reveals the molecular mechanisms underlying specific biological processes and pathogenesis, providing new guidance in disease control and crop improvement (Kell and Oliver, 2016). Additionally, transcriptome studies decipher many dynamic changes occurring in molecular communication during the plant infection by the pathogens. The recent advancement of NGS technologies have allowed the transcriptome sequencing through cDNA sequencing on a massive scale (Voelkerding et al., 2010). This has permitted the researchers to design large-scale experiments to capture and enumerate the transcripts and analyse the transcriptional responses of B. oleracea to black rot infection which revealed the role of different genes involved in B. oleracea-Xcc interaction (Tables 2, 3).

Over the years, transcriptome analysis has been used to understand the plant–microbe interactions (Wulf et al., 2003). Gene regulation studies in response to pathogen attack may indicate the role of the relevant defence genes (Singh et al., 2018). Suppression subtractive hybridisation (SSH) is regarded as a powerful approach for identification of differentially expressed genes including the response of the plants to pathogen infection (Jin et al., 2010; Luo et al., 2010; Yue et al., 2010) which could help in the global analysis of gene expression. SSH does not require sequence information to study differential genes (Kathju et al., 2006) and is often used to identify the genes responding to pathogens and stresses in plants (Xiong et al., 2001). So, Jiang et al. (2011) attempted to understand the molecular mechanisms of resistance of cauliflower in response to Xcc infection combining SSH with RT-PCR. An SSH cDNA library comprising many defence-related genes including plant defensin gene PDF1.2, lipid transfer protein, thioredoxin h., etc. was established and 12 differentially expressed genes associated with Xcc resistance were identified. Roohie and Umesha (2015) also employed the SSH technique to identify the genes involved in black rot resistance mechanisms in B. oleracea var. capitata. Out of 150 unigenes (classified in to five functional categories), 35% of the unigenes accounted for the defence-related unigenes. Defence-specific representation of the genes was confirmed by semi-quantitative RT-PCR and its increased expression in the resistant cultivar was validated by qPCR.

Several researchers have reported the differential responses of genes in the susceptible and resistant cultivars of B. oleracea to black rot disease (Table 3). Cloning of differentially expressed cDNA fragments obtained from black rot resistant cauliflower plants revealed that M6 gene fragment was a new H₂O₂ downstream defence-related gene fragment and could be induced during infection by Xcc (Gu et al., 2008). Transcriptomic analysis of the leaves of B. oleracea collected 3 and 12 dpi revealed the up-regulation of 78 and 809 genes and a downregulation of 10 and 169 genes in the early and late responses, respectively (Tortosa et al., 2018a). During Xcc attack, genes related with terpenes, flavonoids, alkaloids, anthocyanins, SA, ethylene, and JA were up-regulated in early response exhibiting their importance during pathogenesis. To analyse the dynamics of the transcriptional response of B. oleracea plants infected with Xcc, Tortosa et al. (2019) highlighted the role of Ca⁺² signalling proteins as secondary messenger for several downstream signalling processes which include the activation of several transcription factors involved in the SA-mediated host defence in B. oleracea. Two calcium-signalling proteins (CBP60g and SARD1) played important roles in resistance against Xcc which was, again, confirmed by Arabidopsis knockout mutants. In another study, Lee and Hong (2015) analysed the pathogenesis-related (PR) gene expression during resistance and susceptible responses to black rot disease in kimchi cabbage. The semi-quantitative RT-PCR analysis revealed the transcriptional activation of PR1, BGL2, Chi1, PR4, VSP2, LOX2, and GST1 in the leaves of resistant genotypes as compared to susceptible genotypes. Also, the PR genes were found to be regulated by defence-related hormones such as SA, JA, and ethylene. The results indicated that differential defence signalling crosstalk and PR gene expression are involved in cultivar specific resistance against several fungal diseases including black rot in kimchi cabbage and, importantly, the resistance was strongly associated with the hormone-dependent transcriptional induction of defence genes. Jasmonate and other related signalling compounds are involved in the host immunity of plants (Thines et al., 2007; Melotto et al., 2008). A B. oleracea LOX gene was cloned in cabbage which was involved in jasmonic acid biosynthesis, and the quantification of transcript levels showed that BoLOX transcripts were accumulated rapidly after various biotic stresses (Zheng et al., 2007). The JASMONATE ZIM-DOMAIN (JAZ) proteins are the key repressors of jasmonate (JA) signalling and plays key role in plant defence responses (Chini et al., 2007; Thatcher et al., 2016). To investigate the expression difference of cabbage JAZ genes after infection with Xcc, RNA-seq data of both resistant and susceptible materials were analysed by Liu et al. (2020) and many JAZ genes were found to be up-regulated in both the resistance and susceptible lines.

For better resistance against Xcc, it is essential to understand the infection and multiplication process of Xcc in host cells, and tremendous progress have been made in this direction with the identification and characterisation of more than 100 genes contributing to Xcc virulence (Chan and Goodwin, 1999; He et al., 2007; He and Zhang, 2008; Büttner and Bonas, 2010). Liao et al. (2016) investigated the potential role of prc gene in the pathogenicity of the black rot and the transcriptional profiling of the wild type and mutant showed that the mutation of prc in Xcc leads to the alteration of the transcription level of 91 genes. The genes were associated with a range of biological functions such as carbohydrate transport and metabolism, cell wall/membrane biogenesis, post-translational modification, protein turnover and chaperones, inorganic ion transport, metabolism, and signal transduction mechanisms, providing new information about the regulatory role of prc gene. Micro RNAs (miRNAs) are known to be associated with various biological processes including abiotic and biotic stresses (Rubio-Somoza et al., 2009; Kruszka et al., 2012). miRNAs are conserved and are involved in many molecular interaction networks including plant-pathogen interactions (Sunkar et al., 2012; Islam W. et al., 2017). The role of miRNAs in plants was examined in response to Xcc infection and four miRNAs (miR156, miR167, miR169, and miR390) were found to be differentially expressed showing a down and up-regulated expression profile in the susceptible and resistant cultivars, respectively (Santos L.S. et al., 2019). This suggested the possible role of miRNAs in enhancing the resistance of B. oleracea against Xcc and could be used as potential resistance markers for B. oleracea-Xcc interaction. Understanding the early response to black rot holds the key to control black rot and reduce the crop losses. To understand the molecular basis of early-phase response of different resistant cabbage lines against black rot infection, a comprehensive transcriptome analysis of resistant and susceptible lines identified 10,030 differentially expressed genes (Song et al., 2020). Three hundred and eighty four differentially expressed genes overlapped in the susceptible and resistant cabbage lines and the top ten genes contained NBS-LRR type, protein kinase, and expansin genes, indicating their role in early response to black rot infection. Again, transcriptome analysis of the leaves of Xcc-resistant (QP07) and susceptible (DBP71) lines was carried out to understand the early defence response (Sun et al., 2020). A total of 3,357 up and 4,091 down-regulated genes were identified between QP07 and DBP71 and functional annotation pathway analysis indicated the enhancement of the ROS scavenging, glucosinolate biosynthesis and catabolic pathways, hormonal, receptor kinase-related genes, and (NBS)-encoding R-genes during the early infection period. Furthermore, photosynthetic energy metabolism was found to be actively regulated by the host plant in response to Xcc infection. Glucosinolates (GSLs) play important roles in plant defence mechanisms against necrotrophs, biotrophs, and hemibiotrophs and are broadly found in different Brassica species. However, the information about GSL-mediated resistance mechanisms and GSL biosynthesis and catalysis related gene expression after black rot infection is limited. In a recent study, Rubel et al. (2020) found positive and significant association between the aliphatic and indolic GSL compounds with the expression values of transcription factor and GSL biosynthesis-related genes in cabbage. The phytohormones, SA and JA, are the central regulators in hormonal signalling pathways to induce defence response against the pathogens by inducing the genes related to phenylpropanoid synthesis pathway which produces an array of defensive metabolites and these genes were differentially expressed for Xcc resistance and susceptibility in Brassica species. Islam et al. (2019b) demonstrated that the enhanced expression of JA signalling was concurrently based on transcriptional up-regulation of PAP1, MYB transcription factor, and phenylpropanoid biosynthetic genes (CHS, F5H, COMT1, CAD2) which induced the higher accumulation of defensive metabolites such as hydroxycinnamic acids and flavonoids in the resistant cultivar (cv. Capitol). Another study reported the differential expression of the NBS-LRR encoding R-genes (ZAR1 and TAO1) and related genes (MAPK6), calcium signalling-related genes (Ca2+ATPase, CDPK5, CBP60g, CAS, CaM), SA receptor (NPR3, NPR4), synthesis and signalling (ICS1, NPR1) genes, JA synthesis (LOX2), and signalling (PDF 1.2) genes in the contrasting genotypes of B. napus which indicated that JA induced an antagonistic depression of SA suggesting the proper maintenance of SA/JA ratio as a part of the resistance mechanisms against Xcc (Mamun et al., 2020), also proved by Islam M.T. et al. (2017).

Taken together, it could be inferred that genes related to photosynthesis, glucosinolate biosynthesis and catabolism, phenylpropanoid biosynthesis, ROS scavenging, calcium signalling, hormonal signalling and synthesis pathway, receptor-kinase-related genes, and NBS-encoding resistance genes were differentially expressed upon Xcc infection. The examination pattern of hormone-related DEGs revealed that instead of SA signalling pathway, JA signalling pathway may play a critical role in host resistance to hemibiotrophic pathogen such as Xcc. The up-regulation of the genes involved in glucosinolate biosynthesis and catabolic processes during early infection confirmed the role of glucosinolate hydrolytic products in the defence of Brassica species against Xcc. The enhancement of resistance to Xcc by GSLs has been confirmed by several proteomic and metabolomic studies. Several transcriptome studies have revealed the NBS-LLR encoded resistance genes as the key regulators involved in black rot resistance in Brassica species. Also, the transcriptome studies revealed that photosynthesis is playing a major role in the interaction between Xcc and Brassica species. Down-regulation of the DEGs involved in photosynthesis in the resistant plants and up-regulation in susceptible plants suggested that the susceptible plants require more energy to cope up with the infection by Xcc. In contrast, by reducing the photosynthetic metabolism, the resistant plants try to control the energy supply of Xcc, thereby inhibiting its growth. This also shows the greater resilience of the resistant plants in restoring the normal photosynthetic metabolism compared with the susceptible plants. Upon pathogen attack, rapid production of reactive oxygen species (ROS) leading to oxidative burst is described as one of the earliest responses of the host to pathogen infection onsetting the hypersensitive response. However, to adapt to ROS toxicity, the enzymatic and non-enzymatic antioxidants are activated to scavenge the ROS and reduce the oxidative stress which is part of the resistance mechanisms. The up-regulation of the ROS scavenging enzymes such as catalase, superoxide dismutase, glutathione peroxidase 5, and several glutathione S-transferases in the resistant plants indicated that balancing of host oxidative stress response is essential to efficiently control Xcc.

Proteomics is the high-throughput study of total proteins expressed in a particular organism, organ, specific tissue, and cell of an individual in a given time or developmental stage. Proteomics deals with the analysis of protein–protein interactions, protein expression profiles, protein trafficking, localisation, and their various roles in different cellular processes. Unlike the genome of an organism which is relatively fixed, the proteome is highly dynamic similar to transcriptome and changes based on temporal or environmental factors. There are many proteins present in an organism and its presence is dependent on several factors, including the response to abiotic and biotic stress (Renaut et al., 2006). Proteomics is regarded as a tool for functional genomics in plants and serve to analyse major signalling and biochemical pathways and complex responses of plants to environmental stimuli (Setia and Setia, 2008). Protein is the final executors of most of the biological processes translating plethora of genomic information into functional information. Proteomics could be very informative while studying the plant stress response and tolerance either in a genome-wide or sample-scale (Nakagami et al., 2012). Post-transcriptional modifications such as proteolysis, glycosylation, phosphorylation, nitrosylation, and ubiquitination mediate the functions of a large fraction of proteins (Mann and Jensen, 2003; Beck et al., 2006) playing a key role in intracellular signalling, controlling of enzyme activity, protein turnover, transport, cell structure integrity (Wu et al., 2011), and also to understand the molecular mechanisms of plant-pathogen interactions (Quirino et al., 2010; Lodha et al., 2013). Quantitative proteomics could reveal the differentially expressed proteins contributing to stress response process as well (Liu et al., 2015).

Several powerful techniques are used to identify and quantify proteins of complex biological samples. The most widely used methods during high-throughput protein analysis are gel-based techniques (Chevalier, 2010). The quantitative measurement of proteins can be performed using SDS-PAGE, but for identification and characterisation of the separated proteins, two-dimensional polyacrylamide gel electrophoresis (2DPAGE or 2-DE) and mass spectrometry (MS) is required (Eldakak et al., 2013). 2-DE has become the most versatile tool for protein separation as it resolves the proteins based on both isoelectric point (separated according to their pI in pH gradient PAGE) and molecular mass (SDS PAGE, separated according to molecular weight) (Pomastowski and Buszewski, 2014). However, some disadvantages such as labour intensiveness, low reproducibility, insensitiveness to low-copy number proteins, etc. (Eldakak et al., 2013) have hampered the utility of this method. A modified version of 2-DE, difference gel electrophoresis (2D-DIGE), circumvents most of these issues which can control gel-to-gel variation, allows multiple samples to be co-separated, and enhance the reproducibility (Beckett, 2012). Currently, mass spectrometry (MS) is the most commonly used technique for proteome analysis (Aebersold and Mann, 2003). Before MS analysis, the pre-separation of complex protein mixtures is done by 2-DE and cleaved into smaller peptides. Different types of MS methods have enhanced automation in proteome analysis and have replaced the gel-based separation techniques of peptides. Mass spectroscopy includes several approaches such as liquid chromatography–mass spectrometry (LC-MS/MS), ion trap–mass spectrometry (IT-MS), and matrix-assisted laser desorption/ionisation–mass spectrometry (MALDI-MS), etc. (Helmy et al., 2011, 2012; Komatsu et al., 2014; Shao et al., 2015).

Comparative proteome analysis may help in understanding different biotic stresses in B. oleracea as the language of plant-pathogen lies in the proteins. After the first proteome analysis carried out in the model plants Arabidopsis thaliana (Kamo et al., 1995) and rice (Komatsu and Tanaka, 2005), several advances were made in proteomics to answer the complex biological questions. In B. oleracea, proteomic approach was used to understand the mechanisms of interaction of black rot with the host plants (Table 4). Andrade et al. (2008) employed in vivo proteome analysis for protein expression characterisation of Xcc in close interaction with B. oleracea and showed that in vivo expression method originally developed for Xanthomonas axonopodis pv. citri can be successfully employed for Xcc. So, for the first time, in vivo global proteome analysis of Xcc was carried out and the protein profiles of Xcc was compared during the interactions with resistant and susceptible cultivars of B. oleracea (Villeth et al., 2009). The results obtained revealed a group of proteins exclusive to the resistance interaction. Interestingly, different isoforms of the same protein were found in the resistance and susceptible interactions, indicating the same protein playing different roles depending on the types of interaction. The authors also observed the up-regulation of proteins involved in photosynthesis during the resistance interaction.

To identify the proteins involved in pathogenicity, the interaction of Xcc-B. oleracea was studied using an in vivo system in three conditions using the label free shotgun 2D-nanoUPLC/MSE (Santos et al., 2017). A model for Xcc-susceptible host interaction was proposed, which showed that Xcc increases the abundance of proteins required for pathogenicity and cell protection. Pathogenicity related proteins such as acetylornithine (ArgD) and several defence and stress-related proteins (lipoxygenase, annexins, apocitocrome f, antimicrobial compound phytoalexin) was observed in the susceptible (REK) and resistant (REU) Brassica plants, respectively. Also, proteins associated with photosystems were identified in the resistant plants. Importantly, the confirmation of the differential expression of the selected genes indicated that these genes, directly or indirectly, are involved in the Xcc colonisation of the host plant which could be used as future targets for knock-out studies to confirm their role in the pathogenicity. Identification of proteins expressed during plant–pathogen interactions to know which proteins confer disease resistance is essential to understand the plant-pathogen interactions. A novel peroxidase isozyme and lignification in hydathodes were involved in resistance to black rot disease in cabbage (Gay and Tuzun, 2000). Costa et al. (2014) identified 22 differential proteins during early infection by Xcc in B. oleracea. One of the proteins identified was precursor of peroxiredoxin which was decreased in the susceptible genotype, and proteins involved in the photosynthesis were also found to be modulated by Xcc infection which may help in better understanding of the B. oleracea–Xcc interaction.

A study by Vega-Álvarez et al. (2021) has shown that Xcc infection reduces biomass and photosynthesis in the aerial parts of the seedlings though no effect was detected on the leaves or the biomass of the inoculated adult plants of B. oleracea. The biochemical studies state that stomatal closure happens in the presence of Xcc (Ho et al., 2013). Abscisic acid (ABA) is a signalling molecule which can suppress the plant immune response. It has been shown that increased abundance of proteins involved with ABA may favour susceptibility (Kim et al., 2011; Desclos-Theveniau et al., 2012). The ABA signalling pathway is manipulated by a type III effector (AvrXccC8004) of Xcc, thereby increasing the ABA levels (Ho et al., 2013) and proteins responsive to ABA (Santos C. et al., 2019) during the infection by Xcc. ABA along with ROS and elicitors of plant defence may stimulate Ca²⁺ influx (Tortosa et al., 2019) which may increase Ca²⁺ in guard cells prompting stomatal closure (Klüsener et al., 2002). So, stomata can work as part of the innate immunity of a plant by preventing Xcc entry (Gudesblat et al., 2009).

Xcc infection promotes changes in the secondary and primary metabolism in the host to induce defence programs affecting growth and development (Vega-Álvarez et al., 2021). The changes mostly include down-regulation of proteins involved in photosynthesis (Ribeiro et al., 2018; Santos C. et al., 2019). An earlier study (Villeth et al., 2016) had shown a clear reduction in the abundance of proteins involved in energetic metabolism in susceptible interaction with an opposite behaviour in the resistance interaction. This study indicated that resistance to black rot in B. oleracea is correlated with the ability of the plants to keep sufficient photosynthesis metabolism activity to provide energy supplies necessary for an active defence. This was also reported by Villeth et al. (2009). Later, detailed studies of photosynthesis-related proteins upon Xcc attack in Brassica spp. divulged the down-regulation of these proteins in the resistance reactions. Ribeiro et al. (2018) reported the reduction of photosynthesis-related proteins in the susceptible plants of B. oleracea at an early stage of infection with Xcc. Also, decreased abundance of ubiquitin (to stop the bacteria from using ubiquitination pathway) and malate dehydrogenase (to reduce energy metabolism in the early stage of infection) were found, playing important roles in the resistance mechanisms against Xcc. Proteome analysis of Xcc-infected young cabbage leaves and chloroplast-enriched samples of both the susceptible and resistant cultivars revealed the differential abundance of photosynthesis-related proteins in both resistance and susceptible interactions (Santos C. et al., 2019). As expected, most of the photosynthesis-related proteins showed decreased abundance (18%) in the resistance interactions, whereas in the susceptible interaction, increased abundance of proteins was observed consistent with the previous result obtained by Ribeiro et al. (2018). Additionally, proteome analysis revealed the differentially abundant proteins involved in cell metabolism, protein biosynthesis, processing and degradation, and disease/defence response. Among the genes encoding differential proteins, the functional validation of a CHI-B4 like gene encoding an endochitinase showed that the transgenic plants were highly resistant to Xcc compared to the wild type which may assist the future breeding programs targeting at black rot resistance in B. oleracea. Islam et al. (2021) described the down-regulation of photosystem II (PS II)-related proteins in the resistant B. napus cultivar (cv. Capitol) while the PS I proteins, ATP synthase, and ferredoxin-NADP⁺ reductase were up-regulated during the characterisation of the resistance mechanisms in the B. napus–Xcc pathosystem. In the resistant cultivar, the innate immunity-related proteins [Zinc finger SWIM domain-containing 7 isoform X2 (ZFD), glycine-rich RNA-binding GRP1A isoform X1 (GRP1A) proteins and mitochondrial outer membrane porin] were highly enhanced. Also, redox-related proteins (thioredoxin, 2-cys peroxiredoxin, and glutathione S-transferase) were up-regulated in the resistant cultivar with high NADH, ascorbate, and glutathione-based reducing potential. However, the proteins mostly involved in protein degradation, C2 oxidative cycle and glycolysis were highly activated in the susceptible cultivar (cv. Mosa).

In summary, though comparative proteomic analysis is an efficient and powerful approach to understand the defence mechanisms of Xcc–Brassica pathosystem, proteomic studies are still at the infancy stage except a handful of works unlike the genomic and transcriptome studies. Nevertheless, the proteomic analysis identified proteins related to photosynthesis, energy metabolism, innate immunity, ROS production and proteolysis, redox homeostasis, and defence signalling pathways involved in the Xcc–Brassica interaction. Specifically, most of the studies focused on the regulation of the photosynthesis-related proteins as a resistance response in both early and later stages of infection. It is suggested that in Brassica, during resistance interactions, photosynthesis is increased at later stages of infection while it is down-regulated at early stages. So, it was hypothesized that the plant tries to minimize the damage caused by Xcc by regulating photosynthesis-related proteins. Additionally, decreased abundance of ubiquitin and of malate dehydrogenase was hypothesized to play an important role in the resistance mechanisms against Xcc. ZFD and GRP1A proteins were up-regulated in the resistant plants and could be the key regulators of ETI driven innate immunity against Xcc in Brassica species. Also, R-protein-mediated mitochondrial permeability transition (MPT) enhanced the accumulation of mitochondrial outer membrane porin triggering programmed cell death, thereby inducing resistance to Xcc.

Metabolome is the complete set of metabolites present in an organism, organ, tissue, and metabolomics refer to the comprehensive profiling of the metabolome of an organism in a particular moment. Metabolomics lies at the phenotypic end of the omics spectrum, capturing the results beginning with the genome and progressing through the transcriptome and proteome (Liu and Locasale, 2017). Metabolomics is the newest among the ‘-omic’ spectrum and has a broad field to develop. Generally, metabolomics is used in combination with transcriptomics or proteomics to investigate the correlation between metabolite levels and genes or protein expression level (Srivastava et al., 2013). The metabolome mainly consists of primary metabolites (involved in the basic functions of the living cell) and secondary metabolites (play important role in plant defence against pests and diseases, Verpoorte et al., 2007). The field of metabolomics has rapidly grown in the past two decades thanks to the advancement in analytical methods and data analysis allowing the understanding of a vast diversity of metabolites within a given sample. Among several state-of-the-art analytical instruments and separation technologies, nuclear magnetic resonance (NMR), gas/liquid chromatography coupled with mass spectrometry (GC-MS/LC-MS), and capillary electrophoresis/mass spectrometry (CE-MS) are the most widely used tools (Jorge et al., 2016) to capture and quantify a wide range of primary and secondary metabolites. Specifically, GC-MS and LC-MS has become fundamental tools to study the biochemical behaviour of plants exposed to pathogen attack (Arbona and Gomez-Cadenas, 2016) due to their unparalleled sensitivity in quantifying many types of phytochemicals.

In plant pathology, metabolomics deals with the profiling of host plant metabolites in response to pathogen infection which helps in the understanding of the host–pathogen interactions through activation/deactivation of metabolites and related signalling pathways (Castro-Moretti et al., 2020). Upon pathogen attack, plants develop different strategies by modifying gene expression and activating metabolic pathways which may accumulate toxic metabolites, thereby killing the pathogen or limiting the damages. Metabolomics has been used to study plant–biotic stress interactions (Tenenboim and Brotman, 2016). The identification of a wide spectrum of compounds synthesized by the plants in response to biotic stresses provides a better understanding of the regulatory processes underlying stress conditions (Badjakov et al., 2012). Over the years, several authors have evaluated the effects of secondary metabolites, glucosinolates, and its hydrolysis products (GHP), conferring resistance to black rot in Brassica crops (Table 5). In B. oleracea, LC/MS-based metabolite profiling during Xcc1 infection revealed the dynamic metabolic changes occurring in the host cell after 48 h of infection, indicating a complex temporal response (Tortosa et al., 2018b). Furthermore, photosynthesis, alkaloids, coumarins, and sphingolipids were shown to play key roles in the metabolic pathways involved in the infection process. Metabolite profiling of a vast number of compounds present in plants could be accomplished by both targeted (Griffiths et al., 2010) and untargeted metabolomic (Schrimpe-Rutledge et al., 2016) approaches. The untargeted approach deals with both the secondary (polyphenols and carotenoids) and primary metabolites, whereas the targeted approach mostly focused on the identification glucosinolates.

Glucosinolates (GSLs) are the unique components of metabolome (Verkerk et al., 2009) found in Brassica vegetables which are structurally highly diverse and mainly fall into three classes such as aliphatic, indole, and aromatic glucosinolates. GSLs are involved in defence mechanisms against plant pathogens, insects, and nematodes (Bennett and Wallsgrove, 1994; O’Callaghan et al., 2000; Buskov et al., 2002; Serra et al., 2002; Kliebenstein, 2004). Brassicaceae family is mostly dominated by aliphatic and indolic glucosinolates (Fahey et al., 2001; Bekaert et al., 2012). The breakdown products of both aliphatic and indolic glucosinolates by an endogenous myrosinases enzyme (b-thioglucoside glucohydrolases) were found to have anti-fungal and anti-bacterial properties in different crops (Manici et al., 1997; Agerbirk et al., 1998; Brader et al., 2001; Tierens et al., 2001; Stotz et al., 2011; Calmes et al., 2015). Several in vivo and in vitro experiments were conducted to estimate the effect of GSLs and their hydrolysed products (GHP) for disease resistance in Brassica crops. Transgenic A. thaliana with modified glucosinolates profile enhanced the resistance against Erwinia carotovora and Pseudomonas syringae pv. maculicola (Brader et al., 2006). In B. napus, cultivars with higher GSL content showed resistance against Alternaria spp. and Leptosphaeria maculans compared to cultivars with low GSLs (Giamoustaris and Mithen, 1995). The in vitro effect of GHP, especially different isothiocyanates (benzylisothiocyanate, 2-phenylethylisothiocyanate, the isothiocyanate mix and sulforaphane) were found effective against six plant pathogenic bacteria such as Agrobacterium tumefaciens, Erwinia chrysanthemi, Pseudomonas cichorii, Pseudomonas tomato, Xanthomonas campestris, and Xanthomonas juglandin (Aires et al., 2009). Though several research projects were dedicated to elucidate the role of GSLs and GHP against fungal pathogens, fewer studies were conducted with pathogenic bacteria and much less with Xcc (Table 5). Several studies have stated that indolic GSLs are related to the resistance to necrotrophs, biotrophs, and hemibiotrophs in Brassica crops (Hiruma et al., 2013). Xcc being a hemibiotropic pathogen, the role of GHP was evaluated against Xcc infection in various Brassicaceae seedlings and positive correlations were found between specific and total GSL contents and the severity of Xcc infection, though no significant correlations were reported between Xcc infection and total phenolics (Aires et al., 2011). Nevertheless, the susceptibility of the Brassica plants against Xcc was found to be higher in plants with lower contents of aromatic-GSLs and glucoraphanin, both acting as inhibitors of Xcc. This necessitates further requirements of clear and detailed in vitro studies to evaluate the role of GSLs and its GHP in defence mechanisms against Xcc. So, experiments were carried out to evaluate the in vivo and in vitro antibacterial activities of gluconapin, its isothiocyanate (ITC) against Xcc type 4 in B. rapa (Velasco et al., 2013). The results demonstrated gluconapin and its ITC varieties possessing antibacterial effect on the development of Xanthomonas, and gluconapin playing a role in imparting constitutive resistance to Xcc. Additionally, the methanolic extracts of B. rapa containing glucosinolates and phenolic compounds curbed the growth of Xcc. Though GSLs and its hydrolytic products show a wide range of antimicrobial activities, our knowledge on the role of specific metabolites in defending the Brassica crops and their interactions with the pathogens is poorly understood. So, the individual role of two aliphatic (sinigrin, glucoiberin) and one indolic (glucobrassicin) GSLs against Xcc was explored in B. oleracea var. acephala L. (kale) and the indolic GSL glucobrassicin was found inhibitory to Xcc infection than the aliphatic GSLs (Madloo et al., 2019). Interestingly, the results indicated that increasing the amount of a particular GSL may not always result in disease resistance. Instead, its effects are dependent on the pathogen and the type of GSLs. Also, factors like modification of the metabolites during pathogen infection may regulate its affect in inhibiting the pathogens. This is noteworthy as the host genotypes exhibit different GSL profiles and concentrations in different genetic backgrounds.

The variation in GSL contents could happen due to the allelic variation of GSL biosynthesis genes (Rangkadilok et al., 2002). In B. oleracea, three loci (GSL-PRO, GSL-ELONG, and GSL-ALK) were reported to regulate the aliphatic GSL profile (Li et al., 2001). In Arabidopsis, upon fungal infection, the CYP81F2 gene is expressed resulting in the hydrolysis of GSLs by PENETRATION2 (PEN2) and accumulated as indolic 4-methoxy-glucobrassicin in cells (Bednarek et al., 2009). But the information about GSL-regulated resistance mechanisms, expression profiling of GSL biosynthesis genes, and interaction between GSL profiles of black rot susceptible and resistant plants is scanty in B. oleracea. In a recent study, the relative expression of 43 GSL biosynthesis and breakdown-related genes were estimated upon Xcc4 inoculation in cabbage (Rubel et al., 2020). In the resistant lines, nine genes showed consistent expression patterns. Expression values of two (ST5c-Bol030757 and AOP2-Bo9g006240) and five genes (MYB34-Bol017062, MYB122-Bol026204, CYP81F2-Bol012237, CYP81F4-Bol032712, and CYP81F4-Bol032714) showed positive association with aliphatic and indolic GSL compounds, respectively. Finally, four aliphatic (glucoiberverin, sinigrin, gluconapin, and glucoerucin) and four indolic (glucobrassicin, hydroxyglucobrassicin, methoxyglucobrassicin, and neoglucobrassicin) compounds were found positively associated with black rot resistance which may help in elucidating the role of GSL biosynthesis and breakdown-related genes in conferring resistance to Xcc in cabbage.

The phytohormones, such as JA and SA, are important central defence signalling molecules which maintain complex interactions with each other and other hormones such as ABA, cytokinin, gibberellic acid, and indole acetic acid to regulate defence responses (Anderson et al., 2004; Martínez-Medina et al., 2017). The antagonistic interaction between SA and JA and synergistic interaction of JA with ethylene have been well documented in previous studies (Pieterse et al., 1996; Van Wees et al., 2000). So, elucidation of cultivar variation in relation to hormonal status is significant determinant of disease susceptibility and resistance to Xcc. In a study, increased ratios of ABA/JA and SA/JA increased the susceptibility to Xcc in the B. napus cultivars which acted as a negative regulator of redox status and phenylpropanoid synthesis required for inducing resistance (Islam M.T. et al., 2017). With the enhanced expression of SA signalling regulatory gene NPR1 and transcriptional factor TGA1, the ratios of ABA/JA and SA/JA increased with the antagonistic suppression of JA-regulated gene PDF 1.2, leading to higher susceptibility of the cultivars. Similarly, Mamun et al. (2020) also reported the R-gene-mediated induction of JA with an antagonistic depression of SA for ETI response suggesting the importance of maintaining proper SA/JA ratio in conferring resistance against Xcc in B. napus (Figure 3A).

Also, plant phenolics are involved in disease resistance mechanisms in different pathosystems which are synthesized by the phenylpropanoid pathway (Dixon et al., 2002) through the conversion of p-coumaric acid (pCA) into hydroxycinnamic acids and flavonoids (Cartea et al., 2011b). Islam M.T. et al. (2017) reported higher accumulation of flavonoids, hydroxycinnamic acids, and proanthocyanidins in the resistant cultivar (cv. Capitol) by the enhanced expression of the phenylpropanoids biosynthesis-related genes (CHS, F5H, and ANR) upon Xcc infection. Furthermore, expression of these genes were found positively correlated with the enhanced expression of JA-signalling gene PDF 1.2 resulting in the elevated resistance. This clearly demonstrated that JA positively regulates the phenylpropanoid synthesis pathway and JA-signalling pathway is involved in conferring resistance against hemibiotrophic pathogen like Xcc. The pre-treatment of Chinese cabbage genotypes with p-coumaric acid (regulates the phenylpropanoid biosynthesis pathway) alleviated the Xcc symptoms with higher accumulation of hydroxycinnamic acids (ferulic acid and sinapic acid) and flavonoids (EGC and EGCG) both in soluble and cell wall-bound form (Islam et al., 2018). The pre-treatment with p-coumaric acid also enhanced the expression of CHS and HCT genes which regulate the synthesis of flavonoids and hydroxy cinnamic acids, respectively, in the phenylpropanoid biosynthesis pathway. The hormonal regulation of pCA-induced resistance against Xcc was investigated by Islam et al. (2019a) who revealed the enhancement of JA content and expression of signalling genes (COI1 and PDF1.2) in plants pre-treated with pCA in B. napus cultivars inoculated with Xcc. In the Xcc-inoculated plants, a higher accumulation of total hydroxycinnamic acids and proanthocyanidins were reported along with the up-regulation of phenylpropanoids synthesis-related genes in the resistant cultivar. To further investigate if hormonal regulation is associated with the higher accumulation of defensive metabolites synthesized by phenylpropanoid pathway, Islam et al. (2019b) reported that enhanced JA levels and signalling positively regulated the accumulation of phenolic metabolites (pCA, SiA, FA, NA, and EGCG) in the cell wall-bound form and was associated with antagonistic depression of SA, ABA, CK, and IAA-signalling genes in B. napus upon Xcc inoculation (Figure 4). This was accompanied by up-regulation of PAP1 (production of anthocyanin pigment 1)-induced phenylpropanoid biosynthesis genes.

Induced resistance mechanisms are also associated with lignification (Simard et al., 2001) and esterification of cell wall polysaccharides by phenolics (especially with pCA, ferulic acid, and sinapic acid) (Azuma et al., 2005; Cvikrová et al., 2006) under pathogen attack. The enhanced activity of two isozymes of peroxidases, guaiacol peroxidase (GPOD), and coniferyl alcohol peroxidase (CPOD) involved in cell wall cross-linking in the resistant cultivar of B. napus, ultimately induced resistance against Xcc (Islam et al., 2019b).