The field of host plant resistance has traditionally revolved around effector-triggered immunity (ETI) and gene-for-gene resistance. However, it is essential to acknowledge that there are other vital dimensions of functional resistance that merit attention. While ETI and gene-for-gene resistance are well-established and integral components of plant defense mechanisms against pathogens, it is equally valid that other significant aspects of functional resistance can sometimes be overshadowed by the emphasis on these two mechanisms. Recent research efforts have been dedicated to identifying and comprehending novel defense mechanisms that enhance plant resistance against well-known phytopathogens. In a recent study (Singh et al., 2022c), we introduced the groundbreaking concept of the Stem-Physical-Strength-Mediated-Resistance (SPSMR) mechanism. This innovative mechanism has fundamentally reshaped our understanding of plant-pathogen interactions, particularly within the context of the Sclerotinia sclerotiorum-Brassicaceae pathosystem. This study marks a critical milestone in our ongoing research endeavor as we delve into unraveling the intricate molecular underpinnings of the SPSMR mechanism. Our primary aim is to decipher the relationship between stem physical strength and the effective defense strategy against S. sclerotiorum. We seek to uncover the specific molecular mechanisms responsible for driving this unique form of disease resistance. Through a comparative transcriptome analysis between resistant and susceptible genotypes, our objective was to elucidate the molecular foundation of the novel SPSMR defense mechanism. In this paper, we shed light on the valuable insights gleaned from our recent research, emphasizing their implications within the broader context of plant immunity and offering a promising avenue for enhancing crop protection strategies.
Our study's findings unveil substantial disparities between resistant and susceptible genotypes in their responses to S. sclerotiorum infection. These differences in resistance are intricately linked to variations in stem physical strength attributes. Our results consistently demonstrate that the resistant genotype exhibits reduced stem lesion lengths and possesses sturdier stem traits compared to the susceptible genotype across various infection stages. These disparities in stem physical strength attributes appear to substantively contribute to resistance against S. sclerotiorum through intricate physiological and mechanical mechanisms. The robust stem structure of the resistant genotype encompasses several key attributes, including heightened stem density, augmented lignin content, increased stem dry matter content, and elevated stem breaking strength. Collectively, these attributes create an environment that impedes the pathogen's entry, movement, and proliferation within the plant. Increased stem density and lignin content establish physical barriers that effectively deter pathogen penetration. Lignin, a complex aromatic polymer, is renowned for fortifying cell walls and conferring structural rigidity. In the resistant genotype, the elevated lignin content may facilitate enhanced cell wall lignification, thereby hindering the pathogen's ability to breach the plant's defensive barriers (Jha and Mohamed, 2022). Of central importance, the stem's breaking strength, as a quantifiable measure of mechanical resistance to deformation, emerges as a decisive factor in resistance. This strength acts as a bulwark against the mechanical forces exerted by the pathogen during its invasion attempts. The negative correlation between stem breaking strength and lesion length attests to the pivotal role of stem mechanical properties in curtailing infection spread. Additionally, the heightened stem dry matter content contributes to the overall robustness and stability of the plant's architecture, rendering it more formidable for the pathogen to establish a foothold. These findings align with existing insights into plant-pathogen interactions, which underscore the significance of physical barriers in impeding pathogen infiltration (Lee et al., 2019; Kashyap et al., 2021). Our study delves deeper by quantitatively assessing specific attributes and elucidating their individual contributions, providing novel perspectives on resistance mechanisms. Our findings concerning the SPSMR resistance mechanism against S. sclerotiorum in Brassicaceae closely align with the research conducted by Mamo et al. (2021) on Lactuca sativa resistance to Sclerotinia minor. Both studies emphasize the crucial role of stem strength in disease resistance. Mamo et al. (2021) identified a strong negative correlation between stem mechanical strength and the severity of basal stem degradation in lettuce, indicating that genetic factors related to stem strength influence resistance. They also proposed that fortifying stem and crown mechanical strength could be a valuable strategy for breeding disease-resistant lettuce. Our findings support these conclusions, suggesting that strengthening stems may be a means to enhance resistance against S. sclerotiorum in Brassicaceae. Both studies highlight the significance of plant architecture, especially stem strength, in mediating resistance to Sclerotinia infections, implying a common approach for disease control strategies in various host plants.
To explore the molecular underpinnings of the SPSMR mechanism, we conducted a comparative transcriptome analysis on the two distinct genotypes at different infection stages. A consistent trend emerges when comparing the responses of the resistant and susceptible genotypes to S. sclerotiorum at various infection stages. Throughout all infection stages, the resistant genotype consistently exhibits a greater number of differentially expressed genes (DEGs) compared to the susceptible genotype. This disparity in DEG counts underscores the robustness of the resistant genotype's transcriptional reprogramming in response to the pathogen. This trend is particularly evident during the early, mid, and late infection stages. The broader and more extensive transcriptional changes in the resistant genotype suggest a more comprehensive and sophisticated defense mechanism at play, while the susceptible genotype's comparatively limited DEG counts may reflect its challenges in mounting an effective response against S. sclerotiorum. In a prior investigation, Singh et al. (2022d) studied the transcriptional responses of two Cicer arietinum genotypes to the filamentous fungal pathogen Ascochyta rabiei. Their findings demonstrated that the resistant genotype exhibited a higher number of DEGs across infection stages compared to the susceptible genotype. This observation implies a strong and comprehensive transcriptional reprogramming in the resistant genotype, possibly indicating an effective defense mechanism against the pathogen. The gene ontology (GO) enrichment analysis highlights distinct functional trends across cellular component, molecular function, and biological process categories. Notably, the resistant genotype consistently exhibits enriched DEGs within cellular component terms, indicating a more comprehensive cellular restructuring in response to infection. In contrast, the susceptible genotype demonstrates a comparatively less pronounced enrichment in these terms, hinting at a less extensive cellular reorganization. The identification of enriched DEGs related to cellular components in response to infection provides valuable insights into how gene expression changes drive cellular restructuring, potentially affecting various organelles and structures, thereby influencing the cell's ability to mount an effective defense against the infecting agent (Kundu et al., 2019). Temporal distinctions in enrichment profiles indicate divergent strategies. The resistant genotype exhibits heightened gene enrichment across cellular GO terms during early and late infection stages, implying proactive adaptations throughout the infection process. Meanwhile, the susceptible genotype displays increased enrichment during the mid-infection stage, suggesting a targeted response during that specific phase. This correlation in gene expression patterns among defense components aligns with the findings of Gao et al. (2013), who observed similar gene expression patterns in the interaction between potato and Phytophthora infestans. Furthermore, Dobon et al. (2016) also reported similar observations, noting sequential and temporally coordinated waves of gene expression in a host-pathogen interaction between wheat and yellow rust.
Differentiation in molecular functions further underscores their divergence. The resistant genotype shows enriched gene counts in structural molecule and transporter activities, indicating a focus on cellular integrity and transport mechanisms. In contrast, the susceptible genotype emphasizes molecular transduction and transcription regulation, suggesting its efforts to manage signaling pathways and gene expression. Biological process enrichment exemplifies their overall strategies. The resistant genotype displays widespread enrichment across metabolic, cellular, immune, and regulatory processes, reflecting a multifaceted defense. Notably, it exhibits heightened gene enrichment in biological regulation, carbon utilization, and cell killing during early and mid-infection stages, indicating proactive regulation of processes and strategic resource allocation. In contrast, the susceptible genotype's emphasis on molecular transduction and transcription regulation suggests a more directed regulatory response. Overall, the GO enrichment analysis underscores a trend of contrasting strategies. The resistant genotype employs a diverse range of cellular and molecular functions, orchestrating a comprehensive defense against S. sclerotiorum. This involves continuous cellular adaptations, strategic energy management, and dynamic responses. In contrast, the susceptible genotype seems to focus on specific molecular and regulatory aspects (Chen et al., 2013). The KEGG enrichment analysis further highlights a functional divergence between the resistant and susceptible genotypes in their responses to S. sclerotiorum at different infection stages. The resistant genotype consistently exhibits enrichment in pathways associated with metabolic processes, biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, and antioxidant activity, indicating a dynamic response to the pathogen (Kaur et al., 2022). This enrichment is particularly pronounced during the early and late infection stages, suggesting sustained metabolic reprogramming throughout infection. Conversely, the susceptible genotype showcases enrichment in alternative pathways, such as endocytosis, cellular senescence, glutathione metabolism, MAPK signaling specific to plants, and xenobiotic metabolism through cytochrome P450, indicating its focus on regulatory and stress-response mechanisms in reaction to the pathogen. In response to pathogen challenges, plants employ various mechanisms for regulatory and stress-response purposes. Endocytosis aids in internalizing pathogen molecules for immune recognition, while cellular senescence restricts pathogen spread by sacrificing infected cells. Glutathione metabolism counters oxidative stress during pathogen attack. MAPK signaling pathways transmit stress signals to initiate defense responses. Xenobiotic metabolism via cytochrome P450 enzymes detoxifies harmful compounds, possibly including pathogen toxins. These processes collectively enhance plant resilience by facilitating recognition, limiting spread, countering stress, activating defenses, and detoxifying harmful agents (Ghosh et al., 2019).
Temporal variations are also evident, with the resistant genotype displaying heightened enrichment in certain pathways, like zeatin and steroid biosynthesis, during early and late infection stages. This highlights its specific engagement in biosynthetic and signaling pathways at these time points. In the context of plant-pathogen interactions and disease resistance, zeatin, a cytokinin plant hormone, can activate immune responses and contribute to systemic acquired resistance. It can also be manipulated by pathogens for their benefit. Steroids, including phytosterols and steroidal hormones like brassinosteroids, are vital for maintaining membrane integrity and influencing defense-related gene expression. Steroid pathways intersect with jasmonate and salicylic acid pathways, regulating responses against different types of pathogens. The interplay between zeatin and steroid pathways, along with other hormone interactions, forms a complex network that orchestrates plant defense mechanisms, involving immune activation, membrane adaptation, and hormone-mediated signaling to counter pathogenic threats (Zhang et al., 2022). Conversely, the susceptible genotype emphasizes pathways such as apoptosis and glyoxylate metabolism primarily during the early infection stage, revealing its regulatory and metabolic adaptations during this phase. During early infection stages, apoptosis contributes to controlled cell death for containing pathogens, while the glyoxylate metabolism pathway facilitates metabolic adaptations by converting stored lipids into energy sources, both influencing plant defense strategies (Ye et al. 2021). Overall, the KEGG enrichment analysis underscores a clear divergence in the functional strategies of the genotypes. The resistant genotype prioritizes metabolic and biosynthetic processes throughout infection stages, while the susceptible genotype focuses on regulatory and stress-related pathways. These findings provide deeper insights into their distinct responses to S. sclerotiorum infection and contribute to our understanding of their nuanced defense mechanisms. Our findings contrast with those of Wu et al. (2016), who conducted dynamic transcriptomic analyses to investigate varying defense responses against S. sclerotiorum in resistant (R-line) and susceptible (S-line) B. napus. Across 24, 48, and 96-hour post-inoculation periods, the R-line exhibited a higher count of differentially expressed genes with more pronounced fold changes compared to the S-line. A total of 9001 differentially expressed genes were detected in the R-line. These differences in susceptibility and resistance were attributed to significant shifts in the expression of genes linked to pathogen recognition, MAPK signaling, WRKY transcription regulation, jasmonic acid/ethylene signaling pathways, and the biosynthesis of defense-related proteins and indolic glucosinolates.
The findings presented in this study offer valuable insights into the intricate molecular mechanisms governing cell wall reinforcement in response to S. sclerotiorum infection across diverse Brassicaceae genotypes. These findings illuminate the dynamic expression patterns of cell wall-related genes, providing a deeper understanding of how these genotypes employ distinct strategies to fortify their cell walls when confronted with pathogenic challenges. Notably, arabinogalactan protein (AGP) genes emerge as central players, particularly during the early infection stage. In the resistant genotype, there is a pronounced upregulation of genes encoding various arabinogalactan proteins, including fasciclin-like and classical arabinogalactan proteins, while the susceptible genotype fails to exhibit expression in these genes. This discrepancy underscores the resilient cell wall reinforcement mechanisms at play in the resistant genotype, likely contributing significantly to its defense strategy. AGPs, as complex glycoproteins found in plant cell walls, exert influence on cellulose deposition and cell wall structure through their cross-linking interactions with cellulose and other wall components. These interactions are pivotal for forming the cell wall matrix, establishing cross-links between cellulose microfibrils and AGP arabinogalactan chains, impacting cellulose synthesis enzyme activity, and microfibril alignment. Moreover, AGPs also modulate cell wall porosity, mechanical properties, and growth by affecting cellulose arrangement, thus crucially contributing to cell wall integrity and functionality (Lin et al., 2022). Furthermore, the distinct expression patterns of calcium ion-related genes underscore the importance of calcium dynamics in the responses of these genotypes. The upregulation of calcium-dependent protein kinase 16-like, PsbP domain-containing protein, and rhodanese domain-containing protein in the resistant genotype suggests a role for these genes in calcium-mediated signaling pathways. Conversely, the downregulation of EF-hand domain-containing protein in the resistant genotype hints at the complex modulation of calcium-dependent processes. Calcium ion-related genes play a critical role in enhancing cell wall strength by orchestrating various processes involved in cell wall reinforcement. These genes participate in calcium-mediated signaling pathways crucial for sensing and responding to mechanical stress and external challenges. When plants experience mechanical strain, calcium ions are released as secondary messengers, initiating a cascade of events leading to the activation of genes responsible for cell wall modification and strengthening. These genes are also intricately linked to the activation of enzymes like cellulose synthases, which are involved in cell wall biosynthesis and remodeling, as well as callose deposition (Xin et al., 2023; Bundó and Coca, 2016). Calcium ions serve as cofactors for these enzymes, influencing their activity and guiding the precise arrangement of cell wall components such as cellulose, hemicellulose, and pectin. This controlled assembly enhances the overall rigidity and mechanical resilience of the cell wall (Yip Delormel et al. 2019).
The observed upregulation of genes responsible for cell-wall strengthening in the resistant genotype reinforces its active response to pathogenic invasion. The notable upregulation of genes related to xyloglucan endotransglucosylase/hydrolase (XTH), pectinesterase, germin-like protein, cellulose synthase-like protein (CSLs), expansins, and laccases suggests a coordinated effort to modify and strengthen the cell wall structure. In contrast, the lack of expression or downregulation of these genes in the susceptible genotype may indicate its inability to mount an effective cell wall reinforcement response. The collaborative action of these genes plays a pivotal role in reinforcing cell walls through various mechanisms. XTH enzymes reconfigure xyloglucans, altering their cross-linking with cellulose microfibrils and enhancing cell wall properties. By selectively modifying xyloglucan chains, these enzymes enhance cell wall plasticity, extensibility, and strength, enabling plants to adapt to growth, development, and external mechanical forces (Ishida and Yokoyama, 2022). Pectinesterases influence the degree of pectin methylesterification, impacting cell wall rigidity (Levesque-Tremblay et al., 2015). Germin-like proteins could participate in stress responses and cell wall modification, potentially contributing to structural fortification through the hyper-accumulation of H2O2 and the reinforcement of cell wall components (Banerjee et al., 2010). CSLs are involved in the biosynthesis of other important cell wall polysaccharides, such as hemicelluloses and pectins, which contribute to the overall structural integrity of the cell wall (Daras et al., 2021). Expansins play a pivotal role in promoting controlled cell wall relaxation and extension, enabling plant cell growth and expansion by disrupting non-covalent bonds within the cell wall matrix. In contrast, laccases are enzymes responsible for catalyzing lignin polymerization, a process that reinforces the secondary cell wall's structural integrity by creating a rigid network of phenolic polymers. This lignification process, facilitated by laccases, enhances the mechanical strength of plant tissues, making them more resistant to mechanical stresses and environmental challenges (Badstöber et al., 2020; Narváez-Barragán et al., 2022; Blaschek et al., 2023). This concerted action orchestrates dynamic cell wall adjustments, enhancing mechanical strength. The heightened expression of these genes in the resistant genotype signifies a strengthened cell wall defense against pathogens, potentially involving stronger barriers against S. sclerotiorum invasion and reinforcing the plant's structural integrity. Receptor-like kinases (RLKs) and associated proteins contribute significantly to the intricate defense landscape, with distinct expression dynamics in the different genotypes. The specific upregulation of certain genes in the resistant genotype during infection stages indicates its focused activation of receptor-like proteins as part of its defense strategy. In contrast, the lack of expression of these genes in the susceptible genotype implies its compromised recognition and response mechanisms. RLKs and proteins are pivotal for reinforcing cell walls and stem strength. RLKs, as surface sensors, perceive cues like mechanical stress and pathogens, triggering signals that activate downstream responses. These signals prompt proteins like cellulose synthases and pectin methylesterases, aiding cell wall biosynthesis and modification. RLKs also engage with proteins like expansins, enhancing wall extensibility. Additionally, RLKs activate defense pathways, leading to lignin deposition for increased rigidity. This collaboration fortifies cell walls and strengthens stem integrity (Cai et al., 2023; Huang et al., 2018). In the context of this study, the differential expression of these genes could underpin the varying responses between resistant and susceptible genotypes, with the resistant genotype potentially showcasing heightened activation of RLKs and related proteins, resulting in a bolstered cell wall defense against pathogens and improved stem resilience.
The resistant genotype exhibits significant upregulation of phenylpropanoid biosynthesis genes, particularly during mid-infection, aligning with their role in plant defense. This activation of phenylpropanoid pathways serves to counteract pathogens. In contrast, the susceptible genotype downregulates these genes, lacking a comparable defense. These genes are crucial for enhancing cell wall strength by producing phenolic compounds like lignin and flavonoids, which are deposited in the cell wall to provide robustness. Stem physical strength-mediated resistance against S. sclerotiorum benefits significantly from these compounds, as they bolster cell wall integrity and enhance stem strength, creating obstacles for successful S. sclerotiorum invasion (Olivares-García et al., 2020). Peroxidases, essential for bolstering cell wall integrity and fortifying the structural framework of plant stems, participate in diverse processes, including forming lignin and creating cross-links among cell wall elements. Through catalyzing lignin polymerization and aiding the development of lignin-carbohydrate compounds, peroxidases actively strengthen cell walls. Consequently, the existence and effectiveness of peroxidases play a substantial role in augmenting the mechanical strength and structural resilience of plant stems (Mafa et al., 2022; Herrero et al., 2013). The significance of peroxidase genes in relation to cell wall reinforcement and the physical strength-mediated resistance against S. sclerotiorum is evident from the intriguing patterns observed in gene expression results. One particular gene, BjuA006619, responsible for encoding peroxidase, exhibits dynamic behavior. It shows a substantial upregulation during the mid-infection stage, followed by a notable downregulation at the late infection stage in the resistant genotype. This suggests its involvement in early defense responses followed by later regulatory processes. In contrast, two other genes, BjuA018458 and BjuA040532, also encoding peroxidase, consistently exhibit elevated upregulation at multiple infection stages in the resistant genotype. This suggests a consistent role in bolstering cell wall defenses. Another gene, BjuA007044, displays moderate upregulation specifically at the early infection stage in the resistant genotype, emphasizing its role in initial defense activation. Interestingly, the gene BjuA014975, associated with Glutaredoxin-dependent peroxiredoxin, shows remarkable upregulation at the late infection stage in the resistant genotype. This implies a potential role in managing oxidative stress and reinforcing cell wall defenses. Notably, these identified genes show no expression in the susceptible genotype, underscoring their significance in the resistant genotype's strategy to enhance cell wall strength and stem physical resilience against S. sclerotiorum infection.
Shifting focus to other genes linked with key enzymes of monolignol biosynthesis, cinnamoyl-CoA reductase and cinnamyl alcohol dehydrogenase take center stage as key contributors in this process (Pan et al., 2014). For instance, the gene BjuA021656, encoding cinnamoyl-CoA reductase 1-like isoform X1, exhibits noticeable downregulation in the susceptible genotype. This suggests a potential reduction in the availability of precursors for lignin biosynthesis. In contrast, the gene BjuB003565, encoding cinnamoyl-CoA reductase-like SNL6, displays substantial upregulation in the resistant genotype during the mid and late infection stages. This likely contributes to an increased conversion of lignin precursors. Meanwhile, the gene BjuB014599, which encodes cinnamyl alcohol dehydrogenase-6, undergoes noteworthy upregulation in the resistant genotype during the mid and late infection stages. This observation indicates an augmentation in lignin polymerization. Conversely, this gene remains inactive in the susceptible genotype, potentially limiting lignin synthesis. Another noteworthy gene is BjuO006770, responsible for encoding phenylalanine ammonia-lyase (PAL), a key enzyme within the phenylpropanoid pathway. PAL plays a crucial role in reinforcing cell walls by producing lignin precursors, such as cinnamic acid, which are vital for enhancing the integrity of plant cell walls (Zhang and Liu, 2015). This gene is upregulated in the resistant genotype during the early and mid-infection stages, aligning with the reinforcement of cell walls through increased lignin production – a pivotal aspect of physical strength-mediated resistance. In contrast, the susceptible genotype experiences the downregulation of BjuB023238 (phenylalanine ammonia-lyase 2 isoform X1), leading to compromised cell wall strength due to reduced lignin biosynthesis. This mechanistic understanding deepens our grasp of resistance dynamics, shedding light on the molecular intricacies that dictate a plant's ability to fortify its defenses against S. sclerotiorum. Altogether, these findings underscore that the distinct regulation of these genes contributes to variations in lignin deposition and modification, ultimately influencing the strength of the cell wall and the physical integrity of the stem in response to S. sclerotiorum. The resistant genotype's potentially reinforced cell wall defense strategy against the pathogen becomes evident through this comprehensive analysis. S-adenosylmethionine-dependent methyltransferases (SAM-MTs) demonstrate variable expression patterns, underscoring the complexity of their involvement in defense responses. Upregulation in the resistant genotype suggests their contribution to defense-related methylation processes. Conversely, downregulation or fluctuating expression in certain genes indicates intricate regulatory mechanisms that might be compromised in the susceptible genotype. SAM-MTs play a pivotal role in enhancing cell wall strengthening and stem physical strength-mediated resistance against S. sclerotiorum by facilitating the methylation of cell wall components. These enzymes, through the methylation of lignin precursors, contribute to lignin polymerization and deposition, fortifying the cell wall's mechanical integrity. The resulting modifications collectively bolster the cell wall's resilience against mechanical stress and potential pathogen attacks (Baysal et al., 2013; Weng and Chapple, 2010). Overall, the increased SAM-MT expression in the resistant genotype could lead to augmented cell wall modifications, reinforcing its structural strength and enhancing stem resistance against pathogens like S. sclerotiorum.
The expression dynamics of cell wall-associated kinases (WAKs) further highlight genotype-specific responses. The upregulation followed by downregulation in the resistant genotype and specific upregulation during different infection stages accentuate the complexity of WAK-mediated defense responses. The unique regulation of these genes in each genotype underscores their role in shaping defense mechanisms. WAKs play a crucial role in enhancing plant cell wall strength and resilience. Functioning as surface sensors, they detect mechanical stress and pathogenic signals, initiating signaling cascades that activate genes responsible for reinforcing the cell wall. Notably, Yang et al. (2021) demonstrated the significance of cell wall-associated kinases as key regulators in countering Verticillium dahliae in American cotton. Our findings highlight that differential WAK expression potentially underlies distinct responses in resistant and susceptible genotypes. Increased WAK activation in the resistant genotype appears to bolster cell wall defense strategies, leading to reinforced stem strength and hindered S. sclerotiorum invasion. Numerous previous studies have extensively investigated the pivotal role that genes associated with cell wall strengthening play in enhancing the physical and mechanical integrity of plants. Research conducted by Wang et al. (2018; 2020; 2023), as well as studies by Xie et al. (2022), Liu et al. (2021), and Cao et al. (2022), have not only shed light on the significance of these genes in conferring physical strength but have also uncovered their involvement in fortifying resistance within various pathosystems. For instance, the interactions between Triticum aestivum-Ceratobasidium cereale (Wang et al., 2018), Oryza sativa-Xanthomonas oryzae pv. oryzae (Xie et al., 2021), Hordeum vulgare-Blumeria graminis f. sp. Hordei (Chowdhury et al., 2017), and Brassica napus-Sclerotinia sclerotiorum (Liu et al., 2021; Cao et al., 2022) have elucidated the pivotal role of these genes. Notably, Sun et al.'s (2022) study pinpointed a strong connection between cinnamyl alcohol dehydrogenase (CAD) and xyloglucan endotransglucosylase/hydrolase (XTH) and the stem mechanical strength of Paeonia lactiflora inflorescence. Conversely, Wang et al. (2018) revealed wheat caffeic acid 3-O-methyltransferase's affirmative contribution to bolstering wheat resistance against sharp eyespot and enhancing stem mechanical strength, possibly by promoting lignin accumulation. Meanwhile, Xie et al. (2021) findings demonstrated rice's reinforcement of bacterial blight resistance by fortifying the sclerenchyma cell walls of vascular bundles. In another vein, Chowdhury et al. (2017) uncovered that the deposition of heteroxylan in proximity to attempted fungal penetration sites on epidermal cell walls heightens physical resistance against fungal penetration pegs, thereby enhancing pre-invasion resistance during powdery mildew infections in barley leaves. Similarly, the independent revelations of Liu et al. (2021) and Cao et al. (2022) spotlighted genes intertwined with lignin biosynthesis pathways as key players in enhancing stem physical strength, thereby bolstering resistance against S. sclerotiorum in Brassica napus.
Research into the genetic dynamics of disease resistance and plant-pathogen interactions reveals insights into differing defense strategies between resistant and susceptible genotypes. Comparative analysis of gene expression patterns during infection stages highlights significant differences in response. The resistant genotype demonstrates proactive defense, upregulating pathogenesis-related protein genes (e.g., BjuA012943, BjuA030793) notably at 4DAI, showcasing a robust defense response. This emphasizes strategic defense mechanisms against pathogen invasion. Examination of pathogenesis-related protein 5-like genes reveals genotype-specific modulation, with BjuB047318 downregulated in the resistant genotype but upregulated in the susceptible one. This likely contributes to their differing susceptibility. Lipoxygenases are enzymes in plants that generate oxylipins, including jasmonic acid (JA), a hormone pivotal in plant defense against diseases. When plants are attacked by pathogens, lipoxygenases activate and convert fatty acids into hydroperoxides like 13-hydroperoxylinolenic acid (13-HPOT). This transforms into JA, initiating defense responses like antimicrobial compound production and gene activation. JA's effects deter pathogen growth, bolster cell walls, and overall enhance plant resistance. Although effective, JA-mediated responses can interact with other defense pathways, influencing the plant's holistic defense strategy. In essence, lipoxygenases are key players in plant disease resistance by orchestrating the production of JA and activating defense mechanisms that help plants endure pathogenic assaults (Singh et al. 2022e; Thakur and Udayashankar, 2019). Lipoxygenase-2 gene expression adds nuance, with BjuA026845 selectively upregulated in the resistant genotype at 8DAI, emphasizing its role, while BjuA027233 displays distinct behavior, more upregulated in the susceptible genotype at 4DAI, suggesting genotype-specific pathway modulation. In line with our findings, Guche et al. (2021) also underscored the significance of lipoxygenase-mediated resistance against Erysiphe necator in grapevine. Disease resistance genes also vary significantly; RPS5-like protein (BjuA036954) and TAO1 (BjuB035495) upregulate at 8DAI in the resistant genotype, indicating robust resistance pathway activation. Additionally, disease-resistant gene GTP diphosphokinase (BjuB046551) significantly upregulates at 2DAI, showcasing genotype-specific defense mobilization upon pathogen attack. The regulation of intracellular pH emerges as an additional layer of genotype-specific response dynamics. The divergent behavior of genes like Na+/H + Exchanger domain-containing protein cation/H(+) antiporter 6B (NHX6B) (BjuA019498) in the resistant genotype, displaying downregulation followed by substantial upregulation, underscores its strategic involvement in defense-related pH regulation. In contrast, the high expression patterns of genes like Peptidyl-prolyl cis-trans isomerase (PPIase) encoded by BjuA036301 and BjuA035770 in resistant genotype at different infection stages highlight the intricate regulatory role in intracellular pH regulation. In the context of plant-pathogen interactions, the role of specific genes takes center stage in orchestrating defense mechanisms against invading pathogens. Notably, the gene governing NHX6B and PPIase emerge as crucial components in the battle of S. sclerotiorum-Brassicaceae pathosystem. S. sclerotiorum virulence mechanism hinges on the release of oxalic acid within the host, a potent virulence factor pivotal for disease establishment. This acid not only creates an acidic milieu within the host tissue, directly harming living cells, but also hampers the host's antioxidant defense system. Consequently, the activity of cell wall-degrading enzymes escalates, imperiling host cell wall integrity (Hegedus and Rimmer, 2005). To counteract the adverse effects of oxalic acid, the PPIase comes to the fore as a potentially crucial player. This enzyme could function as an intracellular pH homeostatic mechanism, refolding stress-related proteins to activate H + extrusion. This restoration of intra-cellular pH in the invaded host tissue could prove essential for maintaining the functionality of various cellular processes. Additionally, the gene governing NHX6B also play a significant role in maintaining pH balance within the host's cells, enabling the plant to withstand the acidic onslaught caused by oxalic acid. This dynamic role of NHX6B and PPIase in intracellular pH regulation could hold the key to strengthening the host's defense strategies against the virulence tactics of S. sclerotiorum (Singh et al. 2021; Bissoli et al. 2012). These findings collectively accentuate the distinctive strategies employed by the resistant and susceptible genotypes to combat pathogenic challenges.
Overall, apart from the genes responsible for greater stem-physical strength, the resistant genotype surpasses the susceptible one in possessing a variety of genes responsible for the plant-pathogen interaction pathway, disease resistance and pathogenesis-related proteins. Furthermore, it contains genes that regulate intracellular pH, a factor that makes a significant contribution to countering the virulence tactics employed by S. sclerotiorum. These multifaceted mechanisms collectively contribute to establish a robust defense strategy, ultimately augmenting the plant's ability to effectively counteract the pathogen's attack. These insights contribute to a deeper comprehension of host-pathogen interactions and hold the promise of guiding the development of effective strategies to enhance genetic resistance to S. sclerotiorum in Brassicaceae.