Flagellin-derived peptide flg22 from non-pathogenic Pseudomonas fragi Sneb1990 triggers plant immunity and reduces Meloidogyne incognita infestation

Abstract

Background

Biological control is widely recognized for its environmental benefits and has gained increasing attention. The peptide flg22 derived from biocontrol bacteria Pseudomonas fragi Sneb1990 (flg22^Pf) exhibits significant efficacy against Meloidogyne incognita, but its mechanism is still unknown.

Results

In this study, we cloned the full-length flagellin gene from P. fragi Sneb1990 and found that it shares 32.02% sequence identity with the flagellin from non-pathogenic Pseudomonas syringae pv. tomato DC3000 (Pst DC300). Compared to flg22 derived from Pst DC3000 (flg22^Ps), flg22^Pf contains an amino acid substitution at position 19. Growth inhibition assays in Arabidopsis seedlings confirmed that flg22^Pf activates immunity in an FLS2-dependent manner. The Nicotiana benthamiana leaf inoculation experiments indicated that flg22^Pf significantly induces the expression of immune-related genes PTI5 and WRKY7, thereby enhancing resistance against Pst DC3000 infection. Furthermore, tomato treatment with flg22^Pf promoted H₂O₂ production, ROS accumulation, callose deposition, and lignin accumulation. Consequently, this induction of defense responses resulted in suppressed nematode infestation.

Conclusion

Collectively, our results reveal that flg22^Pf from P. fragi Sneb1990 elicits a multi-layered immune response similar to flg22^Ps, leading to an enhanced early immune response in tomato against M. incognita infestation. This study provides a novel plant immune-based strategy for sustainable M. incognita control.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08196-4.

Background

Tomato (Solanum lycopersicum) may be attacked by a wide range of pests and pathogens. Among these, plant-parasitic nematodes, particularly root-knot nematodes (RKNs, Meloidogyne spp.), pose a critical threat to plants [1, 2]. Research on root-knot nematodes management, biological control bacteria has gained growing preference as an eco-friendly and efficacious biological control approach [3–6]. Among biological control agents, endophytic bacteria promote plant growth and activate systemic resistance to enhance immunity, consequently, they have become a significant focus in advancing alternative methods to control plant nematodes [7–9]. We have previously reported that nodule endophytic bacteria Pseudomonas fragi Sneb1990 stimulates tomato immunity through dynamic defense of salicylic acid (SA) or jasmonic acid (JA), thereby inhibiting nematodes, and notably, P. fragi Sneb1990 flg22 (flg22^Pf) triggers tomato immunity against M. incognita [10].

Flagellin (Flg), the core structural protein of bacterial flagella, is involved in motility and host adhesion and is also a potent elicitor of defense responses in diverse plants [11–13]. A conserved 22-amino-acid (flg22) within the N-terminal region of flagellin is recognized by the plasma membrane-resident receptor-like kinases (RLK) receptor complex FLS2-BAK1, which initiates pattern-triggered immunity (PTI) [14, 15]. Flg22 triggers plant immunity by inducing a series of defense responses. These include early cellular events such as calcium influx, reactive oxygen species (ROS) burst, and stomatal closure, as well as downstream responses like callose deposition, nitric oxide production, and the induction of defense-associated genes [16–18].

Previous studies have established that flg22^Pf triggers immunity against M. incognita in tomato [10]. However, the precise molecular mechanisms underlying this resistance have not been fully elucidated. Moreover, whether the immune response triggered by flg22^Pf is consistent with that induced by flg22^Ps (derived from pathogenic Pst DC3000) remains unclear. To address these questions, we cloned the full-length flagellin sequence of P. fragi Sneb1990, compared its structure with that of Pst DC3000, and analyzed the similarities and differences in immune responses between their corresponding flg22 peptides. Our study confirmed that tomato treatment with flg22^Pf triggers effective immunity against M. incognita by priming immune responses, including ROS production, callose deposition, and lignification, which collectively contribute to a significant reduction in nematode infestation. These findings yield significant theoretical insights into the mechanisms of flg22^Pf-induced resistance against M. incognita.

Methods

Plant materials, bacterial strains and nematodes

The M. incognita-susceptible tomato cultivar S. lycopersicum ‘L402’ was obtained from Liaoning Horticulture Seedling Co., Ltd., China. After surface disinfection for 15 min with a 55 ℃ water bath, one seed per pot was sown in plastic pots (9 cm in diameter and 10 cm in height) containing sterilized peat soil (pH: 6.5–6.8; N, P, K ≥ 12 g/kg; water content ≤ 40%; organic content ≥ 40%; Si ≥ 0.3 g/kg) and sand at a volume ratio of 2:1. Seedlings were typically grown at 25 ℃ with 60% relative humidity under a light intensity of 400–600 µmol m^− 2s^− 1 and 14 h photoperiods in a phytotron. Upon reaching 1 month of age, seedlings were used for subsequent experiments. Arabidopsis thaliana wild-type Columbia (Col-0) and the mutant fls2 (SALK_035689) in the Col-0 background, both obtained from the Arashare platform (https://www.arashare.cn/), were surface-sterilized with 75% ethanol and 5% (v/v) sodium hypochlorite. Sterilized seeds were sown on plates containing 1/2 Murashige & Skoog (MS) medium and kept at 4 ℃ for 3 days. Three days after vernalization, the plates were transferred into a growth chamber, and the plants were cultivated under a 16-h photoperiod at 23 ℃, with 50% relative humidity and a light intensity of 100–200 µmol m^− 2s^− 1. After being grown under light conditions for 5–6 days, A. thaliana seedlings were employed for subsequent experiments. Seeds of Nicotiana benthamiana and Nicotiana tabacum were provided by the Nematology Institute of Northern China at Shenyang Agricultural University. Plants were propagated in an artificial chamber. One seed was sown per plastic pot (9 cm in diameter and 10 cm in height) containing equal ratios of sterilized peat soil and sand. The composition of the peat soil was as specified previously. Plants were maintained under a 16-h light/8-h dark photoperiod at 25 ℃, with 50% relative humidity, and a light intensity of 400–600 µmol m^− 2s^− 1. One-month-old tobacco plants were used for the experiments. All plant experiments used seedlings of uniform size and developmental stage, which were randomly distributed among the experimental groups.

P. fragi Sneb1990 is an endophytic bacterium isolated from soybean root nodules and exhibits significant biocontrol efficacy against M. incognita. The method of bacterial strains P. fragi Sneb1990 incubation was carried out in accordance with Wang et al. [10]. Pst DC3000 was cultured at 28 ℃ on King’s B (KB) medium agar plates (20 g/L tryptone + 1.4 g/L K₂HPO₄ + 1% glycerol + 12 g/L agar, pH = 7.2) supplemented with 25 µg/mL rifampicin. After 48 h of growth, the bacteria were harvested, washed twice with ddH₂O by centrifugation for 5 min at 5000 g, and finally resuspended in ddH₂O to an OD₆₀₀ of 0.01 (1 × 10⁶ CFU/mL).

M. incognita was maintained on N. tabacum in pots containing a mixture of peat soil and sand (v/v, 1:1) at 25–28 ℃ in a greenhouse. Nematode egg sacs were carefully hand-picked and collected from the roots with forceps. The collected egg sacs were surface-sterilized with 0.5% (v/v) NaClO for 3 min and rinsed with ddH₂O for 5–6 times. The sterilized egg sacs were then aseptically transferred to a simple chamber for incubation. Two days later, the hatching J2s were collected, counted, and suspended in a 1% carboxymethyl cellulose solution for inoculation.

Structural analysis of P. fragi flagellin and synthesis of flagellin-derived peptide flg22

Genomic DNA was isolated from strains P. fragi Sneb1990 and Pst DC3000 using a previously described method [19]. To amplify the sequence of flagellin genes, primers were designed based on flagellin gene sequences on GenBank (LT629783.1 and NP_791772.1). The primers used in Flg amplification were listed in Table S1. The gene amplification conditions were as follows: 95 ℃ pre-denaturation for 3 min, 35 cycles of 15 s denaturation at 95 ℃, 15 s annealing at 60 ℃, 1 min extension at 72 ℃, an additional extension time of 5 min at 72 ℃, and storage at 4 ℃. The PCR product was ligated into the cloning vector pMD19-T following the manufacturer’s instruction (Takara). Plasmid DNA was isolated from positive clones, and their sequence was verified by Sangon Biotech (Shanghai) Co., Ltd. The sequences were subjected to analysis by DNAMAN (version 9.0.1.116). The sequence identity among P. aeruginosa PAO1 flg22 (flg22^Pa, obtained from the National Center for Biotechnology Information), flg22^Pf, and flg22^Ps was analyzed using CLC Main Workbench (Version 6.8).

The 3D structures of the Flg^Pf and Flg^Ps were generated by homology modeling using the Phyre2.2 server for comparative analysis. To investigate the binding of flg22^Pf or flg22^Ps to the receptor complex, the crystal structure of FLS2-Flg22-BAK1 obtained from the Protein Data Bank (PDB: 4MN8) was used as a reference. Using PyMOL (Version 2.5.1), we performed structural visualization and generated the FLS2-flg22^Pf-BAK1 and FLS2-flg22^Ps-BAK1 complexes by replacing the residues of flg22 with those of flg22^Pf and flg22^Ps, respectively.

The binding affinity of flg22^Pf or flg22^Ps to FLS2 or BAK1 was calculated using the MutaBind2 server [20]. Based on the crystal structure of FLS2-Flg22-BAK1 (PDB:4MN8). The residues of flg22 were substituted for flg22^Pf and flg22^Ps, respectively. ΔΔG_bind values were obtained (-1.5 ≤ ΔΔG ≤ 1.5 kcal/mol means not deleterious).

Peptide sequences of flg22^Pf (TRLSSGLKINSAKDDAAGMQIA) and flg22^Ps (TRLSSGLKINSAKDDAAGLQIA) were custom synthesized at > 95% purity by Sangon Biotech (Shanghai). Both flg22^Pf and flg22^Ps peptides were dissolved in Milli-Q water as 1 mM stock solutions.

Functional evaluation of flg22 peptides in eliciting plant immunity

The growth inhibition assay was employed with minor modifications [21]. Briefly, following a 3-day vernalization and a subsequent 5- to 6-day period under light, A. thaliana seedlings of Col-0 and fls2 were transferred into a 24-well plate. They were treated with 400 µL of 1/2 MS liquid medium containing either 1 µM flg22^Pf or flg22^Ps, with 1/2 MS liquid medium alone serving as the control. The 24-well plates were placed in an incubator for normal cultivation. Each biological replicate consisted of 10 seedlings. After 8–12 days of further growth, the dry weight and root length were measured.

Challenge-inoculation assays for functional PTI evaluation were conducted following Wei’s methodology [22]. Primary treatment involved infiltration of N. benthamiana leaves with 10 µM flg22^Pf or flg22^Ps peptide (upper circles), using ddH₂O treatment as the control. After 6 h, the strain Pst DC3000 (bottom circles) was inoculated overlappingly with an OD₆₀₀ = 0.01 (1 × 10⁶ CFU/mL) concentration. After 3 days post inoculation (dpi), the leaves showing HR response were counted, photographs were acquired, and symptom scoring was performed based on the percentage of necrotic area in the overlapping region. Symptom scoring was conducted in a blinded manner to avoid subjective bias. The assay was carried out with three biological replicates.

Colonization assay

To visualize bacterial colonization, P. fragi Sneb1990 was labeled via electroporation with the shuttle vector pMP2444, which carries genes for gentamicin resistance and the green fluorescent protein (GFP) [9, 23, 24]. Transformants were selected on LB plates containing gentamicin (40 µg/mL), and GFP expression was verified by epifluorescence microscopy (Olympus BX53) using an FITC filter. The gfp-tagged strain Sneb1990 was cultured in LB medium at 28 ℃ with shaking (120 rpm) for 48 h. Bacterial cells were collected (5000 g, 10 min) and resuspended in sterile water to 1 × 10⁸ CFU/ml. Tomato seedlings were irrigated with 10 mL of the bacterial suspension. Each treatment consisted of thirty replicates (one seedling per replicate), and all treatments were maintained under the same growth conditions. Plants were harvested at 1, 3, 7, 14, 21, 35, and 49 dpi. After surface-cleaning three times with sterilized water, samples were sectioned and directly observed under a confocal laser-scanning microscope (Olympus, FV3000, Japan) with 488 nm excitation.

To evaluate the root colonization capacity of the Sneb1990 strain, 0.2 g of fresh root tissue was collected. Following surface sterilization with NaClO solution for 3 min, the samples were thoroughly rinsed with sterile distilled water in three successive 5-min washes. The sterilized roots were homogenized, and the homogenate was serially diluted and plated onto LB agar plates supplemented with gentamicin (40 µg/mL). After incubation at 28 °C for 48 h, bacterial colonization was quantified as colony‑forming units per gram of fresh root (CFU/g) according to Luna et al. [25]. The experiment included three biological replicates.

Evaluation of nematode resistance induced by Sneb1990 and flg22^Pf treatment

To evaluate induced systemic resistance against M. incognita, tomato plants were pretreated with either Sneb1990-derived bacterial preparations or synthetic flagellin peptides (flg22^Pf and flg22^Ps) prior to nematode inoculation. The method of the Sneb1990 fermentation broth was performed according to Wang et al. [10]. Bacterial cells and supernatant were collected by centrifugation at 5000 g for 10 min. The bacterial cell was resuspended in sterile water to obtain a bacterial suspension adjusted to 1 × 10⁸ CFU/mL. Heat-killed bacteria were prepared by incubating the suspension in a 70 ℃ water bath for 1 h. The cell-free supernatant was obtained by filtration through a 0.22-µm membrane filter. Control treatments included sterile water and blank culture medium. Each one-month-old tomato seedling was treated with 20 mL of the respective bacterial preparation (bacterial suspension, heat-killed suspension, or cell-free supernatant) or control solution via root drenching. Twenty-four hours post-treatment, each plant was inoculated with approximately 500 J2s. Nematode infestation was evaluated at 3 dpi.

One-month-old tomato seedlings were root-drenched with 20 mL per seedling with 1 µM of flg22^Pf, flg22^Ps peptide, or Sneb1990 fermentation broth. The control group was treated with the same amount of liquid medium. Twelve days after treatment, root weight and plant height of tomato plants were assessed. Following the same pretreatment method described above, all treatment groups were inoculated with M. incognita after 24 h, and nematode infestation was assessed at 12 dpi.

A nematode inoculation assay was conducted as described with slight modifications [26]. Four holes were made around the roots of tomato seedlings. The holes were inoculated with a nematode suspension containing 500 J2s and then covered with soil. The plants continued to grow in the growth chamber. After the indicated time points, the roots were stained by 3.5% acid fuchsin staining. For 3.5% acid fuchsin staining, tomato roots were decolorized in 5% (w/v) NaClO for 4 min and soaked in distilled water to remove excess NaClO. Roots were then placed in a boiled 3.5% acid fuchsin staining solution, rinsed with water, cooled, dried, and finally transferred to acidified glycerol for documentation using an Olympus microscope. A completely randomized design was used in the experiment. All samples were anonymized with coded labels before counting, and all phenotypic counts were performed by researchers blinded to the treatment groups.

Detection of flg22-induced ROS, callose, and lignin in tomato

To perform the 3,3’-diaminobenzidine (DAB) staining assays, the tomato roots were treated with 1 µM flg22^Pf or flg22^Ps for 6 h. Control treatments included a group pretreated with the kinase inhibitor K252a for 1 h and a ddH₂O group. The treated tomato roots were submerged in DAB solution (1 mg/mL, pH 3.8), vacuum infiltrated slightly for 20 min, then incubated at 25 ℃ for 10 h in the dark, and subsequently transferred to 95% ethanol at 80 ℃ and boiled for 15 min. The seedling roots were rinsed with ddH₂O completely. The dyed seedling root tips were examined and photographed under a microscope. The staining intensity of DAB was quantified with ImageJ software (version 1.54). H₂O₂ content of roots was measured according to the instructions of the Hydrogen Peroxide (H₂O₂) Content Assay Kit (Solarbio, Beijing, China).

For 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) staining to detect ROS, according to the manufacturer’s instructions (MCE, HY-D0940), the treated root tips from the above treatments were incubated at room temperature in 10 µM staining solution for 30 min and washed with ddH₂O three times. The staining results were observed. All images were observed on an Olympus FV3000 confocal microscope (Tokyo, Japan). The quantitative analysis of fluorescence intensity was conducted with ImageJ software.

For the callose deposition assay, the roots of 10-day-old tomato seedlings were treated with 1 µM flg22^Pf or flg22^Ps peptide solution for 10 min under vacuum immersion, and then their roots were cultivated on 1/2 MS medium for 6 h. The ddH₂O and the kinase inhibitor K252a pretreatment served as a control. After 6 h of treatment, the collected root tissues were subjected to vacuum infiltration for 20 min using a fixative consisting of 95% ethyl alcohol and glacial acetic acid (1:1, v/v). The aniline blue staining was carried out in accordance with the manufacturer’s protocol (Solarbio, Beijing, China). Callose deposition was observed using a confocal microscope (FV3000; Olympus, Tokyo, Japan).

The lignified tissues of tomato roots treated with flg22^Pf or flg22^Ps peptide for 24 h were stained using 1% phloroglucinol solution (Xiya, Shandong, China). The kinase inhibitor K252a pretreatment and ddH₂O served as a control. The method was conducted as described with minor modifications [27]. Specifically, the treated tomato roots were immersed in a fixative solution for 24 h. After fixation, roots were rinsed thoroughly with ddH₂O, subjected to vacuum infiltration in a saturated aqueous solution of chloral hydrate for 10 min, and placed at room temperature for 24 h. The transparent samples were soaked in 1% phloroglucinol solution for 5 min, subsequently treated with a few drops of concentrated HCl, and then imaged under a microscope. Lignin was measured using a lignin content assay kit and the colorimetric method (Sangon Biotech, Shanghai, China).

Gene expression analysis by quantitative real-time PCR

Peptides at 10 µM were infiltrated into the leaves of N. benthamiana plants. After 3 h, leaves were collected for RNA extraction. Following irrigation with 1 µM flg22^Pf or flg22^Ps, tomato root tissues were collected at 6 h and frozen in liquid nitrogen. Total RNA was extracted using the RNAiso Plus Trizol following the protocol provided by Takara. Subsequently, RNA samples were reverse transcribed in a 20 µL reaction using the ABScript Neo RT Master Mix for qPCR with gDNA Remover (ABclonal). RT-qPCR was performed with 2× Universal SYBR Green Fast qPCR Mix to determine the relative expression levels of defense-related genes. NbEF1α and Actin gene served as internal reference genes, respectively. Each sample was analyzed in triplicate. Relative changes in gene transcript levels were quantified using the 2^−ΔΔCt method. The primers for RT-qPCR are listed in Table S2.

Statistical analysis

Each experiment included three biological replicates, and statistical significance between two groups was assessed by Student’s t-test, while multiple group comparisons were assessed by one-way ANOVA. The statistical significance analysis was determined with Prism 8.0 software (GraphPad). Differences among the samples were considered significant at p < 0.05.

Results

Comparative analysis of the sequences and structure of flagellin in strains P. fragi Sneb1990 and Pst DC3000

The flagellin of Sneb1990 has a coding sequence of 1680 base pairs (bp), encoding a protein of 559 amino acids (accession: PX682549). Computational analysis using the Expasy server predicted its molecular formula as C₂₃₈₁H₃₉₅₀N₆₉₂O₈₆₀S₈, with a molecular weight of 56.29 kDa, an isoelectric point of 4.94, and a hydrophobicity of -0.123, making it a hydrophilic protein (Expasy, SIB Swiss Institute of Bioinformatics), while the flagellin of Pst DC3000 is a 282-amino-acid full-length protein (accession: WP_005767010.1). Full-length amino acid sequence alignment revealed a low overall identity of 32.02% between Flg^Pf and Flg^Ps (Fig. 1a), indicating substantial divergence. Despite this overall divergence, we found the N- and C-terminal regions, which form the D0 and D1 domains, were found to be highly conserved (Fig. 1b, c). In addition, the D2 and D3 domains showed high variability in both sequence and predicted structure, indicating a high level of polymorphisms in these regions between the two proteins. Comparative analysis of the flg22 epitopes identified that flg22^Pf differs from flg22^Pa at five amino acid positions. Notably, it differs from flg22^Ps by only a single amino acid at position 19 (Fig. 1d).

Fig. 1.

Sequence alignment analysis of flagellin genes and visualization of the Pseudomonas fragi Sneb1990 (flg22^Pf) with the FLS2-BAK1 complex. a Alignment of the nucleotide sequences of flagellin genes of P. fragi Sneb1990 (Flg^Pf) and Pst DC3000 (Flg^Ps). b-c 3D structure of the Flg^Pf and Flg^Ps created by the phyre2 and viewed using PyMOL. d Sequence alignment of flg22 from P. aeruginosa PAO1 (flg22^Pa), Pst DC3000 (flg22^Ps), and flg22^Pf. Alignment is created using the sequence alignment tool of CLC Main Workbench 6.8. e The crystal structure of the Flg22-FLS2-BAK1 complex (PDB: 4mn8). f-g The superimposition of the docked complex (Flg22-FLS2-BAK1 in wheat-cyan-yellow) with the crystal structure 4mn8 (Flg22-FLS2-BAK1 in green-blue-yellow) had similar folding and positioning in the structure alignment. The flg22 (wheat) interacted with the FLS2 (cyan) spanning LRR. The interaction of flg22 (wheat) with FLS2 (cyan) was stabilized through H-bonding (yellow dash) and hydrophobic interaction. The difference between (f) flg22^Ps and (g) flg22^Pf is the amino acid at position 19 (red)

In A. thaliana and many plants, the bacterial flagellin flg22 epitope is recognized by the receptor FLS2. Crystal structures reveal that flg22 binds within a groove formed by 14 LRRs of FLS2, within its C-terminus positioned at the interface between FLS2 and BAK1 (Fig. 1e), thereby acting as molecular “glue” to promote receptor heteromerization, reciprocal activation, and subsequent immune signaling [15, 28]. To explore potential differences in recognition, we performed in silico modeling of the flg22 epitopes from P. fragi (flg22^Pf) and Pst DC3000 (flg22^Ps) bound to the FLS2-BAK1 complex. Structure prediction results revealed only a subtle alteration in a hydrogen bond network at the C-terminus due to the Gln (Q) to Met (M) substitution (Fig. 1f, g) and do not lead to significant conformational changes in the epitope core.

To assess the potential functional impact of this subtle structural variation, we utilized the MutaBind2 pipeline to predict the binding affinity of flg22^Pf with FLS2 and BAK1 (Table 1). The computational predictions showed that the difference in ΔΔG between flg22^Pf and flg22^Ps was less than 0.3 kcal/mol (-0.34 vs. -0.59 kcal/mol). While such energy differences fall below the threshold typically considered significant (|ΔΔG|>1.5 kcal/mol), the consistently more favorable ΔΔG for flg22^Ps, particularly in Phase 2 (-0.88 vs. -1.12 kcal/mol), suggests a possible advantage in receptor complex stability.

Table 1.

Binding affinity between Flg22 and FLS2 or BAK1

Peptide	Sequence		Phase1 (Flg22-FLS2) ΔΔGbind (kcal/mol)		Phase2 (Flg22/FLS2-BAK1) ΔΔGbind (kcal/mol)
flg22_Pa	QRLSTGSRINSAKDDAAGLQIA
flg22_Ps	TRLSSGLKINSAKDDAAGLQIA	-0.59		-1.12
flg22_Pf	TRLSSGLKINSAKDDAAGMQIA	-0.34		-0.88

Letters are underlined indicate the mutated residues compared to flg22_Pa

ΔΔG_bind (kcal/mol): a positive value indicates a destabilizing mutation and a negative value a stabilizing mutation

Pre-treatment with flg22^Pf induces plant immune responses in A. thaliana and N. benthamiana

We evaluated the growth phenotype of A. thaliana seedlings following treatment with 1 µM flg22^Pf or flg22^Ps peptide. The primary root length in the flg22^Pf and flg22^Ps treatments was significantly shorter than the control treatment with 1/2 MS medium (Fig. 2a, b). In addition, the fresh weights of whole seedlings in the flg22^Pf or flg22^Ps peptide treatment were reduced compared to the 1/2 MS control (Fig. 2c). However, the growth inhibition of A. thaliana induced by the flg22^Pf or flg22^Ps peptide was not observed in mutant fls2 (Fig. 2d, e). Although the flg22^Pf and flg22^Ps are distinct from each other as well as from the standard flg22^Pa, they all inhibited the growth of A. thaliana Col-0 seedlings in an FLS2-dependent manner. Consistent with the response to flg22^Ps, this indicated that the perception of flg22^Pf also strictly depends on FLS2 [28, 29].

Fig. 2.

Effects of flg22 peptides on seedling growth inhibition of A. thaliana. a Seedling growth inhibition of liquid 1/2 Murashige-Skoog media containing ddH₂O, flg22^Pf, or flg22^Ps. b-c Quantification of root length and fresh weight per A. thaliana seedling. Different letters indicate statistically significant differences between different treatments (one-way ANOVA; p < 0.05). All experiments were repeated three times with similar results. d Effects of flg22 peptides on growth of Col-0 and fls2 seedlings. The flg22 peptides were administered at a concentration of 1 µM. e Quantification of fresh weight of A. thaliana seedlings. Error bars represent the standard error of 10 biological replicates. Letters indicate significant differences (one-way ANOVA; p < 0.05; ns, not significant)

To investigate the effect of flg22^Pf as an immune response elicitor in eliciting plant immunity. We examined challenge inoculation against Pst DC3000 and measured the expression level of PTI marker genes in N. benthamiana leaves treated with 10 µM flg22^Pf or flg22^Ps. Leaf phenotypes monitored over 72 h showed no chlorosis at 24 h. By 72 h, necrotic lesions developed exclusively in Pst DC3000-inoculated zones, whereas tissues co-treated with flg22 exhibited complete suppression of hypersensitive response (HR). This finding indicated that flg22^Pf treatment may stimulate the PTI of the plant, which inhibited the infiltration of Pst DC3000 (Fig. 3a, b). We used RT-qPCR to analyze the expression of PTI5 and WRKY7 in treated leaves. PTI5 and WRKY7 genes were significantly induced in N. benthamiana leaves treated with flg22^Pf or flg22^Ps (Figs. 3c, d). Notably, flg22^Pf induced a markedly higher expression of PTI5 compared to either flg22^Ps or the control (Fig. 3c). These results confirmed that flg22^Pf can effectively stimulate plant immunity.

Fig. 3.

Plant immune response triggered by flg22 peptides. a Challenge-inoculation HR tests for functional PTI were performed by first infiltrating N. benthamiana leaves with 10 µM flg22 peptides (upper circles). After 6 h, the HR-inducing strain Pst DC3000 (bottom circles) was inoculated overlappingly with an OD₆₀₀ = 0.01 concentration. b HR symptom score grade (0–4). Score criteria: 0 = no HR symptoms; 1 = faint chlorosis, no necrosis; 2 = localized necrosis (< 25% infiltrated area); 3 = extensive necrosis (25%–75% infiltrated area); 4 = complete necrosis (> 75% infiltrated area). c-d PTI marker gene expression induced by 10 µM flg22 peptides in N. benthamiana. Different letters indicate statistically significant differences between different treatments (one-way ANOVA; p < 0.05). All of the experiments were repeated three times with similar results

Exogenous application of the flg22^Pf peptide enhances tomato resistance to M. incognita without affecting plant growth

To investigate the effect of flg22 treatment on tomato biomass and the colonization ability of Sneb1990 in the roots, we measured tomato biomass and monitored bacterial population dynamics. The results showed that neither flg22^Pf nor flg22^Ps significantly affected root weight or plant height compared to the control (Figure S1). Meanwhile, Sneb1990 successfully colonized roots, with its population peaking at 21 dpi before gradually declining (Figure S2). Notably, Sneb1990 inoculation significantly promoted root growth (Fig. 4a and Figure S1b).

Fig. 4.

Pretreatment with flg22 peptides or P. fragi Sneb1990 induces resistance against M. incognita in tomato. a The number of nematodes in tomato root systems irrigation with 1 µM flg22^Ps, flg22^Pf, or Sneb1990 fermentation broth was counted at 12 dpi. Representative images of fuchsin-stained M. incognita parasites on tomato root at 12 dpi. b Nematode numbers per tomato roots at 12 dpi. (c) Ratio of (J3 + J4)/ (J2 + J3 + J4). Bars in the different graphs represent the standard error of the data from three independent biological replicates. Different letters over bars indicate significant differences (one-way ANOVA, p < 0.05)

We then evaluated the role of flg22^Pf in tomato defense against M. incognita. Pretreatment with either Sneb1990 or synthetic flg22 peptides significantly reduced nematode infestation compared to the control without affecting the developmental processes (Fig. 4a-c). Taken together, these results showed that exogenous flg22^Pf application effectively primes tomato resistance against M. incognita without impairing plant growth, an effect comparable to that of flg22^Ps.

The flg22^Pf treatment triggers MTI responses in tomato

To comprehensively understand the role of flg22^Pf in tomato resistance to M. incognita, we measured H₂O₂, ROS accumulation, callose deposition, and lignin accumulation following flg22^Pf treatment. We utilized DAB staining to detect the accumulation of H₂O₂ in tomato root tip tissues and found that the flg22^Pf or flg22^Ps treatments significantly induced the H₂O₂ levels in the root tips (Fig. 5a-c). DCFH-DA staining also detected that the ROS biomass in the root tips of the flg22^Pf or flg22^Ps treatments was higher than that of the control group (Fig. 5d, e). However, there was no difference in the accumulation of H₂O₂ and ROS between the flg22^Pf and flg22^Ps treatments. Compared to the control, callose deposition triggered by flg22^Pf was significantly increased in tomato roots, showing a similar level of accumulation to that induced by flg22^Ps (Fig. 5f, g). Moreover, the lignin staining and content in tomato roots also exhibited a similar result to that of callose deposition (Fig. 5h, i). Further investigation showed that the flg22-induced accumulation of ROS, callose deposition, and lignin was reduced by kinase inhibitors K252a (Fig. 5). Collectively, these results demonstrated that both flg22^Pf and flg22^Ps activate the immune signaling pathway in tomato roots, which orchestrates a multi-layered immune response, including the production of ROS, callose deposition, and lignin accumulation, thereby establishing an integrated defense system in tomato roots.

Fig. 5.

The flg22 peptide treatment triggers immune responses in tomato. a H₂O₂ visualization by DAB staining. Scale bars = 200 μm. b Quantification of DAB staining intensity from (a) using ImageJ. c Quantification of H₂O₂ content in tomato roots. d DCFH-DA staining for ROS in the root tips of tomato after 1 µM flg22 peptide treatments. e Quantification of fluorescence intensity from d. Scale bars = 200 μm. f Callose deposition detected by aniline blue staining, scale bars = 100 μm. g Quantification of callose deposition per field of view in tomato root tips. h The staining of lignin in tomato root with phloroglucinol staining, scale bars = 100 μm. i Quantification of lignin content in tomato roots. Error bars represent the standard deviation. Different letters indicate a significant difference at p < 0.05 (one-way ANOVA)

The flg22^Pf triggers transcriptional reprogramming of defense-associated genes in tomato

Studies have indicated that pathogenic bacteria flg22^Ps can activate the expression of defense genes [30]. In order to further investigate the immune response of tomato plants triggered by flg22^Pf, we analyzed the expression level of genes related to different signaling pathways in tomato root tissues. As described previously, the leucine-rich repeat (LRR) receptor-like serine/threonine-protein kinase FLS2 perceives flg22 to activate defense responses against pathogens [31]. In the present study, treatment with flg22^Pf, the expression of FLS2 was significantly up-regulated (Fig. 6a). To evaluate the role of the biosynthesis and regulation of plant hormones in the induction of tomato root resistance by flg22^Pf. We found that the transcriptional levels of the specific marker genes associated with the SA signaling pathway (PR1, PR2, NPR1) and the JA/ET signaling pathway (PR3, PR4, MYC2) were markedly increased in flg22^Pf-treated tomato roots compared with the control, whereas the expression of PDF1.2 was reduced (Fig. 6b, c). In addition, a hallmark event of response to flg22 is the rapid generation of ROS in plants [32]. Subsequently, we monitored the expression of respiratory burst oxidase homologs (Rbohs) and found that the transcript levels of RbohB and RbohD were up-regulated. This result aligns with the hallmark ROS burst typically observed in flg22-triggered plant immune signaling. In contrast, the expression of RbohF was significantly reduced (Fig. 6d). These transcriptional changes, together with the observed ROS accumulation, suggest that flg22^Pf treatment may activate an Rboh-dependent ROS production pathway. This is consistent with the established role of ROS in early plant defense and may have collectively contributed to the early nematode resistance.

Fig. 6.

The flg22^Pf peptide triggers transcriptional reprogramming of the plant immunity in tomato. a-f Tomato seedlings were treated with 1 µM flg22^Pf. Transcript levels of key defense-associated genes were measured by RT-qPCR. Data are presented as means ± SD (n = 3). *: p ≤ 0.05, **: p ≤ 0.01, ***: p ≤ 0.001, and ****: p ≤ 0.001 by Student’s test

We also examined callose deposition, a well-studied PTI marker in diverse plant species. The expression levels of key biosynthetic genes CalS1 and CalS11 were significantly upregulated (Fig. 6e). Similarly, lignin plays a crucial role in the defense mechanism of tomato plants against root-knot nematodes [33]. We examined the transcriptional levels of key genes involved in lignin biosynthesis to assess their potential roles in the activation of tomato resistance against M. incognita by flg22^Pf. The results demonstrated that the expression of PAL, C4H, and F5H genes was considerably upregulated in the treatment with flg22^Pf (Fig. 6f), indicating that flg22^Pf treatment was involved in the lignin metabolism. Taken together, these data indicated that flg22^Pf activates plant immune signaling pathways, which contribute to enhancing tomato resistance against nematode infestation.

Discussion

Biocontrol bacterium P. fragi Sneb1990, an endophytic strain isolated from soybean root nodules, was previously reported to exhibit efficacy against M. incognita and shows potential as a biological control agent for agricultural production [10]. Preliminary assays indicated that both heat-killed Sneb1990 and its cell-free supernatant exhibited inhibitory activity against nematodes, implicating structural components (MAMPs) and secreted metabolites as contributing factors (Figure S4). Among them, the mechanism by which bacterial flagellin flg22, a key MAMP, activates plant basal immunity to resist pathogen infection has been extensively studied [34–36]. It has been reported that pretreating Arabidopsis with the defense inducer flg22 significantly decreases the invasion by root-knot nematode juveniles [37]. It is worth noting that prior studies have focused on flg22 from phytopathogens, whereas our work characterizes flg22^Pf from the biocontrol bacterium Sneb1990. In this study, we investigated the role of the flagellin flg22^Pf in Sneb1990-induced tomato resistance to nematodes. The results revealed that flg22^Pf from P. fragi Sneb1990 activated both SA and JA/ET signaling pathways, stimulating an ROS burst and inducing callose deposition and lignin accumulation, which collectively contributed to enhanced tomato immunity against M. incognita infestation (Fig. 7). However, the complete biocontrol activity of Sneb1990 is likely attributable to a synergistic interaction between flg22^Pf-mediated immune activation and other contributing mechanisms. Therefore, flg22^Pf is one of the multiple factors through which the biocontrol bacterium Sneb1990 exerts its biocontrol function. It enhances tomato resistance against M. incognita by activating systemic plant immunity.

Fig. 7.

A proposed model for flg22^Pf-induced resistance against M. incognita in tomato. This model illustrates that flg22^Pf peptide activates ROS, callose deposition, lignin, and transcriptional reprogramming of defense-related genes, collectively enhancing root physical and chemical defenses to inhibit nematode infection

As previously reported, flagellin from different bacterial species can have various levels of polymorphism in amino acid sequences [38–40]. The structure and function of flagellin have been intensively studied, which indicates that flagellin has four structural domains: D0, D1, D2, and D3. The research demonstrated that the conserved D0 and D1 domains of Edwardsiella tarda flagellin are sufficient to induce macrophage activation in vitro, despite structural differences in the D2 and D3 domains [41]. Our analysis reveals that despite significant variation in the full-length structure of the Flg^Pf and Flg^Ps flagellin, its D0 and D1 domains are highly conserved. The sequence variation in the flagellin may be the result of the co-evolution of plants and biocontrol bacteria P. fragi Sneb1990 and could facilitate colonization by the biocontrol strain.

Flg22, a conserved 22-amino acid epitope in the N-terminus of flagellin, is recognized by the plant receptor FLS2 and heteropolymerizes with the co-receptor BAK1, initiating signaling and subsequent immune responses [12, 28, 42]. BAK1 plays an important role in signal transduction and innate immunity, and its response to different signal inputs is specific [43]. Previous studies have shown that the first 17 amino acids at the N-terminal of the flg22 epitope, representing the binding sites of FLS2 (address), and the last 5 amino acids at the C-terminal are the binding sites of BAK1 (message) [44]. The amino acid at the 14th position polymorphism of flg22 determined elicitation activity [45]. In line with this, our data show that the amino acid at the 14th position of flg22^Pf is D (Asp), indicating that flg22^Pf can bind to FLS2 and has a stable excitation activity ability. Moreover, the flg22^Pf-triggered immune response was significantly reduced upon co-treatment with the kinase inhibitor, further confirming its dependence on canonical signaling pathways (Fig. 5). Notably, prior studies demonstrated that sequence polymorphisms in flg22, including amino acid variations within or between species, can maintain or differentially modulate its plant immune-triggering capacity [46, 47]. Computational prediction analysis revealed that the amino acid substitution at position 19 in flg22^Pf resulted in a reduction in its binding affinity for the receptor complex (Fig. 1d, Table 1). Our study further revealed that flg22^Pf elicited similar immune responses in A. thaliana and S. lycopersicum compared to flg22^Ps (Figs. 2 and 5), and treatment with flg22^Pf did not inhibit tomato growth (Figure S1). The lack of this growth inhibition may be attributed to its predicted low receptor-binding affinity, thereby avoiding excessive allocation of resources to defense and creating an immune environment conducive to symbiosis. Further experimental evidence of the interaction between flg22^Pf and FLS2 would be an effective approach to explain this phenomenon.

Seedling growth inhibition in Arabidopsis is a robust bioassay for flg22, which reflects a physiological switch from a growth to a defense program. It is demonstrated that a significant correlation has been observed between flg22-eliciting activity and seedling growth inhibition [48–50]. In agreement with a previous study, our experiments confirmed that both flg22^Pf and flg22^Ps similarly inhibit Arabidopsis seedling growth via the FLS2/BAK1 pathway (Fig. 2). Moreover, flg22^Pf treatment stimulates the immune response of N. benthamiana, which hindered the infiltration of Pst DC3000 (Fig. 3). This aligns with the onset of functional PTI in N. benthamiana, which becomes detectable within approximately 6 hpi with avirulent bacterial strains [51].

The flg22 peptide is known to elicit defense mechanisms in plants, leading to various signaling responses, including ROS generation, callose deposition, and production of lignin [52–54]. Notably, flg22 elicits a rapid oxidative burst in plants, which is one of the earliest observable events in plant immunity [12]. In tomato, RbohB (functionally equivalent to RbohD in A. thaliana) is the primary NADPH oxidase responsible for flg22-induced ROS production [55, 56]. Studies have shown that chemical elicitors and beneficial microbes can similarly elevate H₂O₂ levels in tomato roots, and such early oxidative responses are directly associated with reduced nematode infestation [57]. In line with these established mechanisms, our study observed a significant increase in both the flg22-triggered ROS burst and H₂O₂ accumulation (Figs. 5a-e), aligning with the early spatiotemporal ROS pattern critical for nematode resistance as demonstrated in the tomato-M. incognita pathosystem [58]. Correspondingly, qPCR analysis revealed a strong induction in the expression of both RbohB and RbohD genes, but a downregulation of RbohF, upon flg22^Pf perception (Fig. 6d). This differential regulation within the Rboh gene family may reflect their specificity of action in different contexts [59]. However, our data primarily provide correlative support for ROS production and the upregulation of RbohB/D, whereas whether the ROS burst directly depends on Rboh activity and its complete kinetic assay remains to be verified in the future.

Callose deposition is involved in various biological processes. The conserved flagellin domain flg22 epitope was reported to induce callose deposition [60, 61]. Flg22 and nonpathogenic bacteria potently induce callose deposition in roots, demonstrating the first evidence of PTI induction in solanaceous plant roots and serving as a critical early barrier against nematode infestation [30]. In contrast, synthetic Candidatus Liberibacter solanacearum flg22 triggered only weak callose deposition when infiltrated into tobacco, tomato, and potato plants [62]. Based on the above findings, under our experimental conditions, flg22^Pf treatment significantly induced callose deposition (Fig. 5f, g). Accordingly, the transcript levels of the callose synthase genes CalS1 and CalS11 also exhibited a significant increase (Fig. 6e). Furthermore, flg22 enhances the production of defense-related compounds, including the structural polymer lignin [63]. Critically, this reinforcement strategy is known to actively impair nematode infestation. In particular, enhanced lignin content has been shown to directly inhibit the invasion; lignin biosynthesis represents a core component of the plant’s early defense programming, which is triggered prior to the completion of the nematode feeding site establishment [64, 65]. Notably, our data demonstrated that flg22^Pf effectively induced lignin accumulation in tomato (Fig. 5h, i), a core defensive output that fortifies the cell wall. This was further supported by the concomitant upregulation of key genes in the phenylpropanoid pathway, including PAL, C4H, and F5H, which are critical for lignin biosynthesis (Fig. 6f). Interestingly, although flg22^Pf induced lower amplitude early defense gene expression than flg22^Ps (Fig. 6 and Figure S3), it nevertheless effectively triggered robust immune responses in tomato roots, including a ROS burst, callose deposition, and lignin accumulation.

As a well-established early event in PTI, flg22 perception triggers the expression of immune-related genes, often with distinct spatiotemporal patterns [52, 66]. This defense activation is exploited for disease control; for instance, flg22 secreted by Bacillus subtilis OKB105 upregulates PTI5 and SARD1 to enhance immunity [67]. Similarly, flagellin from Pseudomonas putida BP25 elevates the expression of pathogenesis-related (PR) genes in rice (OsPR1.1, OsPR3) [68], and flg22 from Xanthomonas axonopodis pv. dieffenbachiae induces the expression of NPR1 and PR1 genes [36]. However, the root-knot nematode M. incognita employs a strategy by suppressing SA-mediated defenses, as evidenced by the downregulation of PR-1, PR-2, PR-4b, and PR-5 in tomato, which increases host susceptibility [69]. Conversely, analysis of gene expression indicated that flg22^Pf treatment significantly induced the expression of PR1, PR2, and PR4. This suggested that the observed reduction in nematode infestation likely results from flg22^Pf elevating SA pathway activity, thereby counteracting the M. incognita suppression.

Conclusions

In summary, our studies demonstrate that flg22^Pf derived from P. fragi Sneb1990 treatment significantly limits M. incognita infestation in tomato. We found that the flg22^Pf-triggered immune response is similar to that induced by flg22^Ps. Collectively, these findings underscore the potential of P. fragi Sneb1990 as an effective biocontrol bacterium for sustainable nematode control in agriculture. It is important to note that flg22^Pf is one of the biocontrol factors in P. fragi Sneb1990 antagonism against M. incognita, capable of inducing host plant resistance; other active components in the fermentation broth of biocontrol bacteria P. fragi Sneb1990 warrant further research.

Abbreviations

SA

Salicylic acid

JA

Jasmonic acid

ET

Ethylene

Flg

Flagellin

RLK

Receptor-like kinase

PTI

PAMP-triggered immunity

ROS

Reactive oxygen species

PCR

Polymerase chain reaction

PDB

Protein Data Bank

dpi/hpi

days/hours post-inoculation

DAB

3,3’-Diaminobenzidine

DCFH-DA

2’,7’-Dichlorodihydrofluorescein diacetate

HR

Hypersensitive Response

LRR

Leucine-rich repeat

Authors’ contributions

S.Z., wrote the manuscript, with help from L.C., and H.F.; S.Z., and S.W., designed the experiment and analyzed the data; Y.W., XL., N. Y., X.Z., and Y.D. provide technical and intellectual support. All authors have read and approved the final manuscript.

Funding

This research was funded by the National Key R&D Program of China (2023YFD1400400), the National Natural Science Foundation of China (32372481), the Liaoning Province Natural Science Foundation General Program (Grant No. 2024-MS-091), and the National Parasitic Resources Center (abbreviation number: NPRC-2019-194-30).

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.