Natural Elicitor 3,4-Dihydroxy-3-Methyl-2-Pentanone Induces Disease Resistance in Arabidopsis thaliana via Stereoisomer-Specific Activation of Defence Pathways

TL;DR

A natural compound called DMPN can boost plant disease resistance without harming growth, with its four forms activating different immune pathways in Arabidopsis.

Contribution

Discovery of a natural stereoisomeric elicitor, DMPN, that activates plant immunity without growth inhibition.

Findings

DMPN's four stereoisomers induce disease resistance in Arabidopsis without inhibiting plant growth.

Isomers B1 and B2 activate JA/ET and chitin-mediated pathways, while B2 also activates the SA pathway.

Stereoconfiguration significantly affects elicitor activity and pathogen-specific resistance.

Abstract

In contrast to bactericides, elicitors induce plant immune systems to defend against pathogen attack and avoid potential damage to the environment. However, the energy cost caused by the continuous activation of immunity leads to the inhibition of plant growth, which has limited the agricultural application of a large number of elicitors. Here, we identified a natural elicitor 3,4-dihydroxy-3-methyl-2-pentanone (DMPN) that can induce disease resistance in plants. DMPN contains four stereoisomers (3R,4S), (3S,4R), (3R,4R) and (3S,4S), which exhibit different induced resistance activities in Arabidopsis thaliana but do not inhibit plant growth. B1 is different from the other three isomers in that it only induces disease resistance to the necrotrophic pathogen Erwinia carotovora instead of the biotrophic pathogen Pseudomonas syringae, and the remaining isomers is effective for both pathogens. When it comes to threo-isomers B1 (3R,4S) and B2 (3S,4R), transcriptomic and gene expression analysis reveal that both B1 and B2 activated the jasmonic acid (JA)/ethylene (ET) and chitin-mediated signalling pathways. B2 also activated the salicylic acid (SA) pathway and upregulated a wider range of defence-related genes. These findings indicate that stereoconfiguration critically influences elicitor bioactivity. In summary, we reported a natural stereoisomeric elicitor, DMPN, which can elicit the plant defence response in Arabidopsis thaliana without inhibiting plant growth and revealed the differential inducing effects on the plant immune system of its four isomers.

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Full text

1. Introduction

Plants have evolved a system to defend against attack by pathogens, the innate immune system. Plants perceive pathogens by detecting pathogen-associated molecular patterns (PAMPs), activating a plant PAMP-triggered immune (PTI) response [1]. When PAMPs (such as chitin and flagellin) are recognized by cell-surface localized pattern-recognition receptors (PRRs) in plants [2,3,4], a PTI signal is rapidly delivered to the nucleus by cascade reactions, activating transcription factor expression. This elicits a downstream defence response, including the production of reactive oxygen species (ROS), callose deposition and the expression of defence-related genes [5]. Nevertheless, pathogens have evolved another system to cut off the transfer of PTI defence signals through the secretion of specific effector proteins. Effectors may be recognized by nucleotide-binding site-leucine-rich repeat (NBS-LRR) proteins in plant cells, activating effector-triggered immunity (ETI), an amplified version of PTI that induces hypersensitive cell death if the intensity of the signal passes a threshold [2]. Subsequently, defence signals are transmitted from individual cells to the whole plant, mediated by the plant hormones salicylic acid (SA), jasmonic acid (JA) and ethylene (ET), inducing system resistance.

Innate immunity can be primed by biotic or abiotic stimuli [6]. Defence priming is a unique physiological state that can be induced by molecular patterns of microbes or plants, pathogen-derived effectors, beneficial microbes, treatment with some natural or synthetic compounds and by wounding [7]. Primed plants show fast and/or strong activation of defence responses when subsequently challenged by microbes, insects, or abiotic stress [8]. In contrast to direct activation of defences, defence priming is postulated to be a low-cost defensive measure because defence responses are only slightly and transiently activated by priming stimuli [9]. The SA mimic 2,6-dichloroisonicotinic acid (INA) was the first synthetic compound shown to elicit defence responses [10]. Several years later, benzothiadiazole (BTH) was reported to activate defence priming in both cell cultures and intact plants [11]. However, although INA and BTH can both strongly elicit plant systemic acquired resistance (SAR), their practical agronomic use has been limited because they strongly suppress plant growth. β-Aminobutyric acid (BABA), a natural product of plants, is a well-known priming activator that induces broad-spectrum disease resistance in a wide range of economically important crop species [12,13]. BABA has a stereospecific induction capacity since only the R-enantiomer is active in plants. Luna et al. [14] reported that the IMPAIRED IN BABA-INDUCED IMMUNITY 1 (IBI1) gene plays an important regulatory role in BABA-induced resistance. Recently, azelaic acid (AZA) and pipecolic acid (Pip) were reported to be involved in defence priming in Arabidopsis thaliana (A. thaliana). AZA induces the plant to accumulate higher SA levels upon bacterial challenge. Mutation of the AZA INDUCED 1 (AZI1) gene led to specific loss of bacterial- and AZA-induced priming of SA accumulation and attenuated the SAR response [15]. Pip-induced defence priming was also associated with SA, but each acted independently [16].

Many elicitors have been identified from natural or synthesis products. However, most studies have focused on finding a new structure and have neglected the effects of stereoisomerism. Many elicitors have been found to be affected by stereoisomerism, which can significantly impact bioactivity. For example, (2R,3R)-butanediol triggered induced systemic resistance (ISR) against Erwinia carotovora ssp. carotovora SCC1 in Nicotiana tabacum, but another stereoisomer, (2S,3S)-butanediol, did not have the same effect [17,18]. Similarly, only the (R)-enantiomer of BABA can bind IBI1 to encode an aspartyl-tRNA synthetase (AspRS) and trigger immune signalling, not the (S)-enantiomer [14]. 3,4-Dihydroxy-3-methyl-2-pentanone (DMPN) is a natural elicitor of secondary metabolites of the plant growth-promoting rhizobacterium Bacillus subtilis HN09 [19]. In a previous study, DMPN’s threo-isomers and erythro-isomers were isolated from the Bacillus subtilis strain HN09, and they showed different activities in inducing disease resistance [20]. Studies have suggested that the stereoconfiguration critically influences the binding affinity of a compound to its receptor, thereby modulating its bioactivity. Understanding the functional roles of stereoconfigurations in resistance induction enables the targeted biosynthesis of biocontrol bacteria, leading to increased accumulation of effective configurations and improved biocontrol efficacy [21,22,23].

Here, we elucidated the influence of stereo structure on the plant disease resistance induced by DMPN. We synthesized and separated DMPN’s threo-isomers and erythro-isomers, which were named B1 and B2, B3 and B4, respectively. These compounds induced disease resistance differently in A. thaliana. The RNA-Seq results indicated that the chitin signalling pathway was associated with this process.

2. Results

2.1. B1, B2, B3 and B4 Induced Resistance to Pathogens in Plants

We tested the induction of resistance activity among DMPN stereoisomers against the biotrophic pathogen Pseudomonas syringae pv. tomato DC3000 in A. thaliana plants. Four-week-old A. thaliana Columbia (Col-0) plants were treated with B1, B2, B3, B4 and benzothiadiazole (BTH, as a positive control) for 24 h and then inoculated with DC3000. The symptoms of the B1-treated plants were the same as those of the water control, whereas the symptoms of the B2-, B3- and B4-treated plants significantly decreased (Figure 1a). A. thaliana plants treated with B2, B3 and B4 presented significantly (P < 0.05) reduced growth of the pathogen DC3000 in leaves (Figure 1b). Compared with the water control, the density of DC3000 decreased by 50%, 66% and 67% in the plants treated with B2, B3, and B4, respectively, although the positive control BTH decreased the density to a greater extent (Figure 1b). The growth of DC3000 in liquid medium containing 100 μM of B1, B2, B3, or B4 or the commercial fungicide thiram was monitored (Table 1). The four stereoisomers slightly reduced DC3000 growth, whereas thiram completely eliminated the growth of two kinds of bacteria. These data suggest that DMPN protected A. thaliana from DC3000 through a mechanism different from direct toxicity to the pathogen.

Callose is a β-1,3-glucose polysaccharide that is used by plants to strengthen the cell wall to isolate fungal hyphae and stop their colonization and development in the plant tissue [24]. Several reports have demonstrated the relevance of callose deposition in the priming of plant immunity [25,26]. Accordingly, we examined B1-, B2-, B3- and B4-induced callose deposition in A. thaliana Col-0 plants. At 24 h post-infection (hpi), callose deposition in the B1-, B2-, B3- and B4-treated A. thaliana leaves was apparent but was not observed in the CK treatment (Figure 1c,d). At 48 hpi, these defence responses were observed in the CK group, but callose accumulation was significantly greater in the B1-, B2-, B3- and B4-treated plants (Figure 1c,d). These results revealed that when plants were challenged by inoculation with DC3000, all four compounds promoted faster and stronger defence responses, especially B3 and B4.

We also examined DMPN stereoisomer-inducing resistance activity against the necrotrophic pathogen E. carotovora subsp. carotovora strain ECC. As shown in Figure 2a, compared with the water control, the four compounds showed stronger activity in inducing A. thaliana resistance against ECC. Interestingly, the B1 isomer failed to elicit resistance to DC3000 but strongly induced the defence against the necrotrophic pathogen ECC. Notably, the positive control synthetic elicitor BTH strongly inhibited plant growth, whereas B1, B2, B3 and B4 successfully induced plant disease resistance but did not inhibit plant growth (Figure 2b,c), suggesting promising prospects for commercial application as inducers of plant immunity. The proportions of A. thaliana leaves with disease severity level IV or higher in treatments B1 and B2 accounted for 3.33% and 11.11% of the total investigated leaves, respectively, whereas in treatments B3 and B4, the proportions were 17.78% and 34.56%, respectively. These results indicate that B1, B2, B3, and B4 can induce resistance in A. thaliana against the necrotrophic pathogen ECC, but the effects of B1 and B2 are more pronounced.

To evaluate whether the elicitor effect of DMPN is broad-spectrum, we further tested the elicitor’s activity in the rice cultivar Lijiangxintuanheigu, which is susceptible to rice blast disease. Five days after inoculation with Magnaporthe oryzae (GD00-193), rice plants treated with B1, B2, B3, B4, and benzothiadiazole (BTH) exhibited varying degrees of disease symptoms. Among them, the lesions of the rice treated with B1, B2, and B3 were relatively severe, similar to those in the water control. In contrast, plants treated with B4 and BTH also developed lesions but displayed better overall growth (Figure S1a,b). To further investigate these defence responses, we analysed the expression of two disease resistance genes, OsAOS2 (allene oxide synthase 2-like, LOC4332121) and OsLOX (lipoxygenase 7, LOC4345993). Compared with the control, all four compounds significantly increased the expression of these genes (Figure S1c). Although the B1, B2, B3 and B4 treatments did not induce excellent resistance of rice against the fungus Magnaporthe oryzae, these compounds could act as elicitors to activate the rice defence response.

2.2. PR1 and PDF1.2 Gene Expression in B1-, B2-, B3- and B4-Treated A. thaliana Plants

B1, B2, B3, and B4 showed little or no antibacterial activity but appeared to act through immune system activation in A. thaliana in response to the pathogen. The JA/ET signalling pathway plays an important role in the plant immune response to necrotrophic pathogens, and the SA signalling pathway is important in the response to biotrophic pathogens [27]. Consequently, we speculated that the SA, JA and ET signalling pathways could be involved in DMPN-elicited disease resistance. With respect to the SA and JA pathway marker genes AtPR1 (PATHOGENESIS-RELATED GENE 1) and AtPDF1.2 (PLANT DEFENSIN 1.2), AtPR1 was significantly upregulated in B2-, B3- and B4-treated plants compared with the water control, with increases of 10.88-, 9.68- and 5.44-fold, respectively (Figure 3a). As expected, B1 (1.42-fold) failed to markedly upregulate AtPR1 gene expression. However, the expression of the JA-dependent gene AtPDF1.2 was significantly upregulated by 3.99-fold in the B1 treatment. Similarly, B3 and B4 upregulated AtPDF1.2 expression by 2.57- and 9.10-fold, respectively.

We also tested the expression of the AtPR1 and AtPDF1.2 genes in B1-, B2-, B3- and B4-pretreated A. thaliana 24 h after inoculation with DC3000. AtPR1 was significantly upregulated in B2- and B4-treated plants, and AtPDF1.2 gene expression also increased in the B3 treatment (Figure 3b). The different levels of defence responses between the treatments at the same concentration indicates that their ability to induce resistance was affected by their different structures. AtPR1 and AtPDF1.2 appear to be highly responsive genes for priming and excellent indicators of the priming response in plants [28]. The upregulated expression of plant defence-related genes after treatment with the four compounds in response to challenge with DC3000 confirms that DMPN is a strong elicitor of induced plant defence.

2.3. B1-, B2-, B3- and B4-Induced Resistance in jar1, etr1 and npr1 Mutants

To further confirm that JA, ET and SA are involved in the DMPN-triggered plant defence response, we tested whether B1, B2, B3 and B4 induced resistance against DC3000 in jar1 (MeJA-insensitive), etr1 (ET-insensitive) and npr1 (SA-insensitive) mutant A. thaliana plants. Compared with the water control plants, the plants pretreated with B2, B3 and B4, but not B1, presented a significant (P < 0.05) reduction in pathogen growth (Figure 4). However, the observed 40% reduction in this parameter in Col-0 plants was significantly (P < 0.05) greater than the approximately 30% reduction in jar1, etr1 and npr1 mutant plants. A possible explanation is that induced resistance in B2-, B3- and B4-treated plants against DC3000 was partly lost in JA-, ET- and SA-insensitive mutants. Indeed, the classical view often emphasizes the antagonistic relationship between SA and JA pathways in plant immunity, particularly against biotrophic pathogens like DC3000. However, our results indicating that B2-, B3-, and B4-induced resistance to DC3000 was partially compromised in jar1, etr1, and npr1 mutants suggest a more integrated and cooperative role of SA, JA, and ET signalling in the resistance mechanisms elicited by these DMPN isomers. Hormone pathways can act synergistically or additively in certain contexts. For example, overexpression of GmbZIP19 in Arabidopsis enhanced resistance to DC3000, accompanied by upregulation of both JA-related (AtLOX4) and SA-related (AtNPR3) marker genes [29]. This supports the notion that coordinated activation of JA and SA pathways may contribute to improved defence against DC3000. In our study, B2, B3, and B4 likely engage a broad-spectrum defence network that involves SA, JA, and ET signalling.

2.4. B1-, B2-, B3- and B4-Induced Transcriptional Changes

A key step towards a systems-level understanding of the mechanism of the DMPN-induced defence-related response is to obtain comprehensive and accurate insight into the dynamic transcriptional reprogramming that takes place in plants following DMPN stimulation. In this study, we used RNA-Seq technology to profile whole-genome transcription in A. thaliana leaves at 1 h, 6 h and 12 h following the application of B1, B2, B3 and B4. The four isomers induced a large number of differentially expressed genes (DEGs), which were selected according to their significance in terms of fold change expression [false discovery rate (FDR) < 0.01] and an additional threshold level of at least twofold change (|log_2_ (foldchange)| > 1) in comparison to the CK controls. The RNA-Seq results revealed that the number of DEGs increased with increasing treatment time. At 1 h, approximately 300 DEGs were induced by B1, B2, B3 and B4, and the number increased fourfold to approximately 1200 at 6 and 12 h. The number of upregulated and downregulated genes was similar at 1 h and 6 h, but more genes were downregulated at 12 h (Figure 5a). Among the observed DEGs, we selected 9 upregulated genes and quantified them by qRT-PCR to obtain results similar to those of the RNA-Seq analysis (Figure 5b), supporting the results of RNA-Seq analysis (Figure S2). We then analysed the expression patterns of key defence-related genes, including the immune marker gene PLANT CADMIUM RESISTANCE 1 (PCR1), the JA/ET pathway gene AtPDF1.2, NON-RACE-SPECIFIC DISEASE RESISTANCE 1/harpin-induced 1-LIKE (NHL) family gene AtNHL10, the salicylic acid receptor gene NONEXPRESSER OF PATHOGENESIS-RELATED GENE 1 (NPR1), and PATHOGENESIS-RELATED GENE 1 (PR1), PR2, PR4, and PR5. At 1 h post-treatment, AtPCR1, AtNPR1 and AtPDF1.2 were upregulated in A. thaliana leaves treated with B1, B2, B3 and B4. After 6 h, only B2 induced the expression of additional defence-related genes, including AtPDF1.2, AtNHL10, AtPR1, AtPR2 and AtPR5. At the 12 h time point, the expression of most genes returned to levels comparable to those of the control (CK) (Figure 5b). Overall, these results indicate that all the treatments (B1, B2, B3, and B4) successfully activated the expression of defence-related genes in A. thaliana. Notably, B2 induced a broader set of defence genes, which may explain its significant effectiveness against both necrotrophic and biotrophic pathogens.

Through GO enrichment analysis, a dynamic transcriptional level change induced by DMPN was revealed. A greater number of DEGs were enriched for plant immune-related GO terms (Figure 5c–e). At 1 h, DEGs that were induced by B1, B2, B3 and B4 were mainly enriched in innate immune-related GO terms, including “responses to chitin”, “MAPK cascade”, “response to jasmonic acid”, “defence response” and “system acquired resistance”. B1 had stronger activity intensity in terms of “response to chitin (GO:0010200)” than the other three isomers did (Figure 5c and Figure S3). At 6 h, the changes in nuclear activity were significant. Many transcript-related GO terms, such as “histone H3-K9 methylation”, “regulation of DNA replication” and “DNA-templated transcription, elongation”, were enriched. (Figure 5d). Notably, at 12 h, many DEGs were enriched for plant metabolism-related and defence-related GO terms (Figure 5e). These included “response to auxin”, “xylan biosynthetic process” and “cell wall macromolecule metabolic process”. Many DEGs associated with cell wall metabolism were also detected. Moreover, hormone-related GO terms, such as “response to JA” and “response to ET”, were enriched among the DEGs at all time points, indicating that these hormones play important roles in defensive signal transduction. In summary, DNMP-induced DEGs were significantly enriched in defence-related GO terms, which indicates that DMPN can successfully activate the immune system of plants. It is important to note that the four DMPN configurations take different amounts of time in relation to functional dynamics. B1-induced DEGs were more active at 1 and 12 h than at 6 h, B2-induced DEGs were most active at 6 h, while B3- and B4-induced DEGs were more active at 6 and 12 h. Overall, B1 appeared to have the fastest functional response, followed by B2, B3 and B4.

Venn analysis further showed a specific overlap among the DEGs induced by B1, B2, B3, and B4. At 1 h, 107 overlapped in B3 and B4, accounting for 50.47% of the total differential genes induced by B3. B3 coinduced only 70 and 49 genes with B1 and B2, respectively, accounting for 33% and 23%, respectively (Figure S4a). This finding indicates that compared with the B1 and B2 isomers, the B3 isomer triggered an immune response pattern that was more similar to that of B4. This trend became more obvious with time. At 6 h, B3 and B4 coinduced 596 DEGs, while only 173 and 240 DEGs, respectively, were coinduced with B1 and B2 (Figure S4b). At 12 h, B3 and B4 jointly induced 798 DEGs, accounting for 68.44% of the total DEGs induced by B3, which was significantly greater than the corresponding number for B1 and B2 (Figure S4c). Although there was significant overlap in the DEGs induced by B3 and B4, a large difference in the number of DEGs induced was observed for B1 and B2. Combining the previous resistance phenotype and DEG GO enrichment data, we can conclude that the B3 and B4 configurations had similar effects on the inducing of the A. thaliana immune response, whereas the activity of the other two configurations, B1 and B2, significantly differed.

2.5. Configulations of Threo-Isomers of DMPN

Our previous research revealed that, compared with the erythro-isomers (B3 and B4) and a mixture of these four isomers, the threo-isomers (B1 and B2) of DMPN had significantly greater biocontrol activity against the pathogen [20]. However, the results of the current study revealed that B1 and B2 induced different resistances of A. thaliana to pathogens, suggesting the possibility of a different mechanism of inducing disease resistance among the chiral configurations. On this basis, we focused on the configurations of B1 and B2. The configurations were unambiguously assigned by X-ray crystallographic analysis. Compounds B1 and B2 formed colourless rhombohedral crystals. Single-crystal X-ray diffraction analysis revealed that both compounds crystallize in the chiral orthorhombic space group P2_1_2_1_2_1_ (with detailed refinement parameters provided in Table S1), and the asymmetric unit of each compound contained two molecules. For the B1 compound, the unit cell parameters are a = 7.76715(19) Å, b = 7.93346(16) Å, c = 49.176(2) Å, α = β = γ = 90°, and V = 3030.26(17) Å^3^. For the B2 compound, the unit cell parameters are a = 7.7348(3) Å, b = 7.8945(2) Å, c = 48.9077(11) Å, α = β = γ = 90°, and V = 2986.43(16) Å^3^. Structurally, the two compounds display opposite chiral configurations, forming a pair of enantiomers. B1 was determined to have the 3R,4S-configuration, and B2 was determined to have the 3S,4R-configuration (Figure 6a).

2.6. Chitin Pathway-Related B1-Isomer-Primed Plant Immunity

The B1 isomer with a 3R,4S-configuration plays a specific role compared with the other three stereoisomers of DMPN, with the B1 isomer only inducing resistance to the pathogen ECC, and not to DC3000, in A. thaliana. RNA-Seq results revealed many DEGs in response to the B1 treatment at the 1 h time point (Figure 6b). Pathways and GO enrichment analysis revealed that the DEGs induced by B1 were markedly enriched in the “plant–pathogen interaction” pathway and “response to chitin” GO term (Figure 6c,d). Chitin, which is a polymer of N-acetyl-D-glucosamine (NAG), is a well-studied PAMP that can be recognized in A. thaliana by a lysin motif receptor kinase (LYK) [30]. AtLYK5 is the primary receptor for chitin and forms a chitin-inducible complex with AtCERK1 to induce plant immunity [31]. RNA-Seq and qRT-PCR results revealed that, compared with B2, B3 and B4, AtLYK5 expression was significantly upregulated by B1 in a shorter time period (1 h) (Figure S2). Moreover, transcriptomic enrichment in “MAPK cascade” and “respiratory burst involved in defence response” to downstream signal transduction are consistent with canonical chitin signalling (Figure 6d) [30]. B1 also upregulated the expression of the early immune marker gene PCR1 induced by chitin and WRKY family genes (WRKY8, AT5G46350; WRKY26, AT5G07100; WRKY28, AT4G18170; WRKY48, AT5G49520; and WRKY65, AT1G29280) (refer to “original data-Figure 5a.xlsx” file). These findings suggested that B1 engaged components of the chitin-mediated immune response, which further induced MAPK phosphorylation and ROS bursts, after which WRKY family proteins exerted their transcriptional regulatory functions, ultimately leading to disease resistance [32]. AtCERK1 is a receptor-like protein involved in responses to many factors, including chitin signalling. To preliminarily explore the potential interaction between AtCERK1 and the B1 isomer, we performed molecular docking analysis. The hydroxyl group of B1 formed a hydrogen bond with Ser-184 of AtCERK1, with a bond distance of 2.70 Å, while the carbonyl group of B1 established another hydrogen bond with Ser-35 of AtCERK1, with a bond distance of 3.0 Å. Additionally, the hydrophobic methyl group of the compound engaged in hydrophobic interactions with Leu-86, Val-168, Tyr-170 and Val-210 residues of the protein. (Figure S5). Furthermore, molecular dynamics (MD) simulations assessed by root-mean-square deviation (RMSD), root-mean-square fluctuation (RMSF), radius gyration (Rg), solvent accessible surface area (SASA), hydrogen bonds number, and free energy decomposition analysis confirmed the structural stability of the docked complexes AtCERK1-B1 (Figure S6). Taken together, our data revealed that a chitin-related signalling pathway plays an important role in B1 (3R,4S)-induced plant immunity.

3. Discussion

Plant immune elicitors are potential environmentally friendly biological pesticides that rely on activating the plant innate immune system to defend against pathogen attack. Unlike traditional pesticides, plant immune elicitors do not have the risk of developing pathogen resistance. Many elicitors, including some natural compounds, such as BABA and AZA, have been identified [15,33]. Here, four chiral isomers, B1, B2, B3 and B4, of the natural elicitor DMPN (from Bacillus subtilis HN09 secondary metabolites [19,20]) were obtained to further study the mechanism underlying the inducing of plant defence response by DMPN. We evaluated plant protection against two different types of pathogens, namely, biotrophic DC3000 and necrotrophic ECC, in A. thaliana treated with B1, B2, B3 and B4. The results revealed that the B2, B3 and B4 isomers resulted in disease resistance to both DC3000 and ECC, whereas B1 only triggered a specific defence against ECC (Figure 1 and Figure 2a). JA and ET play important roles in plant responses to necrotrophic pathogens, whereas SA is recognized as the key player in responses to biotrophic pathogens [34,35]. We analysed the expression of the JA/ET-inducible gene AtPDF1.2 and the SA-inducible gene AtPR1 in A. thaliana plants at 24 h after treatment with B1, B2, B3, or B4. The data revealed that these two defence-related genes were upregulated to different degrees in response to the B2, B3 and B4 treatments. However, B1 only induced the expression of the JA/ET-related gene AtPDF1.2 (Figure 3). The data from mutant plant experiments also supported these results. B2-, B3- and B4-induced resistance to DC3000 was significantly weakened in jar1 (MeJA-insensitive), etr1 (ET-insensitive) and npr1 (SA-insensitive) mutant A. thaliana plants compared with wild-type plants (Figure 4). Furthermore, in rice, the expression of the JA/ET pathway defence-related genes OsAOS and OsLOX was also upregulated by the B1, B2, B3, and B4 treatments (Figure S1). On the basis of the above results, we propose that B2, B3 and B4 isomers trigger plant defence responses via the JA, ET and SA signalling pathways. For B1, the SA-related GO term “salicylic acid biosynthetic process” was transiently enriched in B1-induced DEGs at the 1 h time point (Figure 5c). However, on the basis of the qRT-PCR (Figure 3) and mutant phenotype (Figure 4) results, the selective activity of the B1 isomer against necrotrophic pathogens is closely tied to its ability to consistently activate JA/ET-dependent defence pathways while showing limited involvement with SA-dependent pathways. The limited activation of the SA-mediated pathway by B1 likely explains its lack of efficacy against P. syringae. This selective pathway activation reduces energy costs and prevents unnecessary immune activation, potentially preserving plant resources for growth and development.

The RNA-Seq results revealed a dynamic change in transcriptional levels upon application of B1, B2, B3 and B4 (Figure 5). Compared with the water control treatment, B1, B2, B3 and B4 resulted in the expression of a large number of DEGs, mainly those related to plant disease resistance, cell metabolism and secondary metabolic product synthesis. Examples included the response to plant disease resistance-related hormones such as SA, JA and ET; the defence response to pathogens; the biosynthesis of plant disease resistance-related hormones such as SA, JA and ET; and disease resistance signalling pathways such as SAR and ISR. Moreover, we revealed that the expression of some key defence-related genes was upregulated by B1, B2, B3 and B4, including AtPCR1, AtNPR1 and AtPDF1.2 (Figure 5b). The above results suggest that DMPN successfully activated the plant innate immune system.

In addition to immune-related GO terms, a large number of DEGs were characterized by GO terms related to DNA transcription, such as “DNA replication (GO:0006260)”, “DNA-templated transcription, elongation (GO:0006354)” and “histone H3-K9 methylation (GO:0051567)”. Chromatin modification is a heritable epigenetic change, and many studies have shown that histone modification is a memory for priming passed to the next generation [36,37]. Keren et al. reported that histone H3K4 and H3K36 methylation was associated with transgenerational priming induced by BABA and INA in the common bean (Phaseolus vulagris L.) [13]. Histone H3K9 methylation was also found to regulate defence mechanisms and flowering time in A. thaliana [38,39]. Chromatin modification is associated with a permissive state of gene transcription. The 6 hpi data revealed a DMPN-induced immune signal transmitted to the nucleus that increased gene expression. At 12 hpi, there was a remarkable enrichment in DEGs associated with metabolism- and development-related GO terms, including “response to auxin”, “cell proliferation” and “meristem initiation”. Elicitors induce plant defence at the expense of energy consumption, which causes plant growth inhibition. As a result, many DEGs were associated with plant growth regulation-related GO terms, such as “response to auxin”. Furthermore, we also noticed that cell wall activity was increased and that many DEGs were enriched in “secondary cell wall biogenesis”, “cell wall macromolecule metabolic process” and “xylan biosynthetic process” GO terms. The cell wall plays an important role in plant resistance to pathogens [40,41]. Cell wall hardening and deposition can disrupt pathogen infection. Xylan is an important component of the plant cell wall, and changes in xylan structure significantly impact disease resistance [35]. The results at the 12 h time point suggest that the defence inducing signals had been transferred to the downstream immune system response and activated corresponding defence responses, such as enhanced cell wall metabolism. Surprisingly, there was no negative effect on plant growth for any isomer of DMPN. In contrast, the plants in the BTH treatment group were significantly inhibited (Figure 2b,c). Maintaining constant activation of the plant immune system requires energy [42]. Many outstanding elicitors, such as INA, BTH and BABA, have limited practical application in agriculture because of decreased crop growth [8,43]. The results obtained here suggest that DMPN does not have this drawback, supporting its potential application value in agriculture.

The chirality of compounds plays an important role in biological activity, but this feature has been mostly ignored in the research on elicitors. Unlike the erythro-isomers (B3 and B4), which exhibited overlapping effects on immune activation, the threo-isomers (B1 and B2) displayed distinct disease resistance-inducing profiles. The timing and intensity of B1-induced gene expression differ from those of other isomers. B1 rapidly activated early immune markers, including genes involved in the chitin response and MAPK signalling cascades, within 1 h of treatment (Figure 5c and Figure 6b–d). Chitin is a representative fungal microbe-associated molecular pattern (MAMP) molecule that triggers various defence responses in both monocots and dicots [44]. When MAMP is recognized by PRRs anchored on the cell membrane, resistance signalling is transferred downstream via the mitogen-activated protein kinase (MAPK) cascade [2]. The molecular docking results further suggested a potential affinity between B1 and AtCERK1, a key component in chitin-mediated immunity (Figures S5 and S6). This fast and targeted activation of defence-related genes ensures a quick immune response to necrotrophic pathogen attacks, further highlighting the functional specificity of the B1 isomer. In contrast, B2 demonstrated superior and broader-spectrum disease resistance-inducing activity. It effectively induced resistance against both necrotrophic and biotrophic pathogens, likely due to its capacity to engage multiple signalling pathways (SA, JA, and ET), and activated a broader range of defence-related genes, including AtPDF1.2, AtNHL10, AtPR1, AtPR2, AtPR5 (Figure 5b). Collectively, these findings demonstrate that B2 was an optimal configuration, combining superior elicitor potency with broad-spectrum resistance, thereby offering high application potential in plant disease management.

4. Materials and Methods

4.1. Materials

B1, B2, B3, and B4 were obtained by WuXi AppTec (Shanghai, China). The Arabidopsis (A. thaliana) mutants jar1 (CS9723), etr1 (CS3070) and npr1 (CS3724) were derived from the SALK mutant library. For soil culture seedlings, A. thaliana wild type Col-0 and mutants seeds were soaked in cold water overnight at 4 °C and then planted in pots with peat soil. Two-week-old seedings were transferred into new pots and grown under fluorescent lights (10 h of light/14 h of dark, 23 °C, 100 μEm^−2^s^–1^). The challenging pathogen Pseudomonas syringae pv. tomato DC3000 was grown in liquid King’s medium B (KB) (20 g/Lpeptone, 1.5 g/L MgSO_4_, 1.5 g/L K_2_HPO_4_, and 10 mL/L glycerinum) containing 50 mg/L of rifampicin at 28 °C overnight. Cultured DC3000 cells were pelleted by centrifugation (2000× g, 3 min), washed once with sterile water and resuspended in 10 mM MgSO_4_ containing 0.01% (v/v) of the surfactant Silwet L-77 (Sigma), and then were adjusted to OD_600_ = 0.01 for inoculation. Erwinia carotovora subsp. carotovora ECC was streaked onto TSA plates (10 g/L tryptone, 10 g/L sucrose, 1 g/L glutamic, adjust pH to 6.8) and incubated at 28 °C in the absence of light for 24 h. The concentrations of E. carotovora strain ECC were adjusted to OD_600_ = 0.01 for inoculum preparations.

4.2. Plant Inoculation and Sample Processing

Phenotypes of four-week-old A. thaliana plants were observed. After spraying the entire leaves of A. thaliana with B1, B2, B3, B4, and BTH (positive control) at a concentration of 100 μM, pathogen inoculation was performed 24 h post-treatment. For ECC, plants were inoculated by injecting bacteria (diluted to OD_600_ = 0.02) into leaves from the back stoma. Plants were then cultured at 100% humidity in darkness and then the diseased area of leaf was recorded after 2 days. For DC3000, inoculum (OD_600_ = 0.02) was sprayed on A. thaliana leaves uniformly. After inoculation, all plants were kept in a dew chamber at 100% relative humidity for 3 days and were then transferred to a growth chamber (10 h of light/14 h of dark, 23 °C, 100 μE m^−2^s^–1^) to show symptoms. Quantification of Pst DC3000 followed the method described by Naznin et al. [45]. To determine the number of Pst DC3000 cells in inoculated leaves, we collected and weighed all leaves from the samples, rinsed them thoroughly in sterile water, then homogenized them in sterilized distilled water. Leaf suspensions were plated on King’s agar supplemented with rifampicin (50 mg/L), and after 48 h incubation at 28 °C, the number of CFU of Pst per gram of leaves was calculated. The experiment was repeated 3 times. Mean comparisons were conducted using a least significant difference (LSD) test (P = 0.05). Standard errors and LSD results were recorded.

An in vitro bacteriostatic experiment was performed to verify whether B1, B2, B3 and B4 had direct antibacterial activity against DC3000. A single colony of DC3000 was selected and incubated in King’s liquid medium at 220 rpm and 28 °C for about 6 h until the logarithmic growth phase. The DC3000 bacterial solution was transferred to the 96-well plate (200 μL per hole) and mixed with 50 μL of B1, B2, B3, B4, Thiram or BTH (500 μM). OD_600_ in each hole was detected immediately after dosing, then the bacterial solutions were cultured at 150 rpm for 24 h and measured for OD_600_ again.

To evaluate the elicitor activity of DMPN against rice blast fungus, a testing assay was performed using the rice cultivar Lijiangxintuanheigu, which is susceptible to rice blast, and the rice blast fungus Magnaporthe oryzae strain GD00-193. When rice seedlings reached the 4–5 leaf stage, the leaves were sprayed with 100 μM solutions of compounds B1, B2, B3, B4, or BTH (positive control). Twenty-four hours after treatment, the rice plants were sprayed with a cell suspension of GD00-193 at 1  ×  10^5^ CFU/mL. Following inoculation, the rice plants were kept in darkness under 100% relative humidity for 24 h to facilitate infection, then transferred to a greenhouse under normal photoperiod conditions (14L:10D). Humidity was maintained by regular misting to promote disease development. Five days post-inoculation, disease symptoms were photographed and recorded for each treatment.

4.3. Callose Deposition

To determine callose deposition, A. thaliana leaves were sampled at 24 and 48 hpi and treated as described in a previous report [46]. Briefly, two or three leaves were first immersed in a destaining solution (phenol/glycerol/lactic acid/water/ethanol (v/v/v/v/v) = 1/1/1/1/8) and vacuum-infiltrated for 10 min to ensure thorough penetration of the solution. The leaves were then incubated in a 60 °C water bath for 30 min to clear the chlorophyll. After rinsing with water, the chlorophyll-free leaves were soaked in 0.01% (w/v) aniline blue stain solution containing 150 mM K_2_HPO_4_ (pH = 9.5) in the dark for 2 to 4 h. Finally, the callose deposition of leaves were observed and photographed using a Leica TCS SP5 X Laser Confocal Microscope (Leica, Germany) at 405 nm excitation wavelength and emission wavelength at 485 nm. The quantification of callose deposition area was calculated by Adobe Photoshop 2015 CC.

4.4. Gene Expression Analysis

Plant samples were wrapped in aluminium foil and rapidly frozen in liquid nitrogen. The snap-frozen samples were then used for total RNA extraction. Total RNA was extracted following the instructions provided with the E.Z.N.A.^®^ Plant RNA Kit (OMEGA, R6827). To verify whether the extracted RNA was suitable for subsequent qRT-PCR analysis, RNA quality was assessed by gel electrophoresis. Due to the propensity of RNA to form secondary structures, denaturing agarose gel electrophoresis containing formaldehyde was employed, which provides an accurate reflection of RNA integrity. The RNA loading mixture (20 μL) for formaldehyde denaturing gel electrophoresis consisted of 2.0 μL 10× FA buffer, 7.5 μL formamide, 2.5 μL formaldehyde, 2.0 μL 10× loading buffer, 3.2 μL DEPC-treated water, and 2.0 μL RNA sample. The mixture was thoroughly mixed, heated at 65 °C for 5 min, then rapidly chilled on ice to eliminate secondary structures. Prior to loading, 0.8 μL of ethidium bromide (EtBr, 1.0 mg/mL) was added to the RNA sample rather than to the gel, reducing background fluorescence during electrophoresis. The prepared 1.2% formaldehyde denaturing gel was pre-electrophoresed in 1× formaldehyde gel electrophoresis buffer for 15 min. RNA samples were electrophoresed at 5–10 V/cm for 30 min. RNA integrity was evaluated based on the band pattern; typically, plant RNA exhibited five bands, and quality criteria included clear 26S and 18S rRNA bands without degradation, with the 26S band visibly approximately 1–2 times more intense than the 18S band. cDNA synthesis was performed according to the PrimeScript™ RT reagent Kit with gDNA Eraser protocol (TaKaRa, RR047A). For qRT-PCR primer design, gene CDS sequences were retrieved from the NCBI database (http://www.ncbi.nlm.nih.gov/), primers were designed using Primer Premier 5.0 software, and their specificity was confirmed through NCBI BLAST analysis.

For the qRT-PCR, cDNA was obtained from RNA samples using the Prime Script RT reagent Kit (TaKaRa, Dalian, China) with oligo dT primers. qPCR amplifications were performed from 10 ng of cDNA using iTaqTM Universal SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) in 96-well plates according to the manufacturer’s recommendations. The real-time PCR was performed on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, CA, USA). AtUBQ5 (825398) was used as housekeeping gene for relativizing expression. Primers used are described in Table S2.

4.5. RNA-Seq Analysis

Four-week-old A. thaliana plants were treated by spraying with 100 μM B1, B2, B3, B4 and MeJA (Sigma, Shanghai, China) solution which contained 0.015% (v/v) Silwet L77 (Sigma, Shanghai, China). The mock solution only contained 0.015% (v/v) Silwet L77. At 1 h, 6 h and 12 h after treatment, the sixth leaf (counted from the oldest true leaf toward the youngest leaf) was harvested from individual A. thaliana plants and snap frozen in liquid nitrogen from three plants for each treatment. RNA-seq was completed by the Hengchuang Gene Technology Company (Shenzhen, Guangdong, China). Sequencing libraries were generated using NEBNext^®^ Ultra™ RNA Library Prep Kit for Illumina^®^ (NEB, MA, USA) following the manufacturer’s instructions. Differential expression analysis was performed using the DEGSeq, q value (or FDR) < 0.01 and |log_2_ (foldchange)| > 1 were set as the threshold for significantly differential expression. Gene Ontology (GO) enrichment analysis of differentially expressed genes (DEGs) was implemented with GO seq, in which gene length bias was corrected. GO functional analysis provides GO functional classification annotation for DEGs as well as GO functional enrichment analysis for DEGs. Enriched GO terms regarding the biological function were based on the Gene Ontology database (http://www.geneontology.org/).

4.6. Molecular Docking and Molecular Dynamics Simulation of AtCERK1 with B1

AlphaFold3 was employed to predict a high-resolution 3D structure model of the AtCERK1 protein (AT3G21630). The structure was subsequently processed and optimized using the Protein Preparation Wizard module in Schrödinger. As it was an apo structure without a bound ligand, potential binding sites were identified using the SiteMap tool. The top-ranked pocket, designated as “Site 1,” was selected for subsequent docking study. Receptor grid generation was then performed using the Glide module, with a grid box encompassing the entire binding site and centred at the coordinates X = −18.619, Y = −3.522, and Z = −23.649. Then, ligands B1 and B2 were prepared using the LigPrep module to generate correct 3D conformations and appropriate protonation states at physiological pH. Molecular docking was performed using the Glide module in Extra Precision (XP) mode. The resulting docking poses were ranked based on Glide GScore, and the top-scoring conformation for each ligand was selected for detailed interaction analysis.

Molecular dynamics (MD) simulations were performed using GROMACS 2024. The AMBER99SB-ILDN force field was applied to the protein [47], while water molecules were described using the SPCE model [48]. Long-range electrostatic interactions were treated using the Particle Mesh Ewald (PME) method [49]. The system was neutralized by adding counterions, followed by energy minimization using the steepest descent algorithm for 5000 steps with a constraint force constant of 2.0 kcal/(mol·Å^2^). Subsequently, the system was equilibrated in two phases. First, a 100 ps restrained NVT simulation was conducted using the V-rescale thermostat to reach 300 K with a weak constraint of 1.0 kcal/(mol·Å^2^). Second, a 1 ns restrained NPT simulation was performed using the Parrinello–Rahman barostat to adjust the pressure to 1 atm. Coupling parameters for temperature and pressure were set to 0.2 ps [50]. Production MD was run for 20 ns under NPT conditions with an integration time step of 2 fs. Covalent bonds involving hydrogen were constrained using the SHAKE algorithm. The non-bonded interaction cutoff was set to 8.0 Å. Trajectories were saved every 2 ps for subsequent analysis. The RMSD and Rg were calculated to evaluate system stability. The final equilibrated trajectory was used to calculate binding free energy with the gmx_PBSA tool.

5. Conclusions

In this study, we reported a natural elicitor, DMPN, that can prime a plant immune system without negatively affecting plant growth, and revealed the different bioactivities of its four stereoisomers. This configuration-dependent bioactivity provides a valuable reference for the practical application of DMPN in agriculture. Our data suggest that all four isomers triggered defence-related gene expression. Regarding the threo-isomers, B1 (3R,4S) rapidly activated genes related to JA/ET signalling pathways and chitin response, while B2 (3S,4R) engaged a wider array of genes linked to SA, JA, ET, and chitin-mediated defence. Moreover, B2 formed a more stable and stronger binding complex with the chitin receptor, supporting its superior elicitor potency. These results provide important information for research on novel plant immune elicitors and their synthesis.