A soybean susceptible variety, Williams 82 (W82), were grown at 27°C with a 12 h photoperiod in a greenhouse. Soybean cyst nematode (Heterodera glycines Ichnohe, SCN) race 3, one of the most widely distributed races in China, was tested in this study. SCN infested soil was mixed 1:1 with sterilized fine sand. Soybean seeds were sown to make SCNs grow and multiply. The cultivation lasted for two months, SCNs were separated from the infested soil, and the eggs were collected and incubated in Baermann Funnels at 27°C avoiding light. The hatched second stage juveniles (J2s) were collected daily and new distilled water was added. In order to ensure the activity of J2s, the collection process will be completed within one week. The isolated J2s SCN were utilized to infect various soybean materials.

For CPT pretreatment, disinfected Williams 82 soybean seeds were coated with various concentrations (9.6 mmol·L^-1, 4.8 mmol·L^-1, 2.4 mmol·L^-1, 1.2 mmol·L^-1, 0.6 mmol·L^-1, 0.3 mmol·L^-1, 0.15 mmol·L^-1, and 0.075 mmol·L^-1) of CPT working solution and placed in plug trays to promote germination. Once the soybeans reached the two-leaf stage, they were transplanted into culture bowls of the same size for further growth. Each soybean plant was then inoculated with 1000 second-stage juveniles (J2) of the soybean cyst nematode. Fourteen days post-inoculation, nematode populations were statistically analyzed using acid fuchsin staining to determine the optimal concentration of CPT solution for inducing soybean resistance to SCN (Cook et al., 2012; Melito et al., 2010).

Total RNA was isolated from soybeans roots using the Total RNA Extraction Reagent (Vazyme, Nanjing, China), first-strand cDNA was synthesized using the PrimeScript™ RT reagent kit (TaKaRa, Beijing, China). qRT-PCR was performed to detect gene transcript levels using the 2X Universal SYBR Green Fast qPCR Mix (ABclonal, Wuhan, China) on a CFX96 qRT-PCR detection system (Bio-rad, San Francisco, CA, USA). Data were analyzed using the 2^-ΔΔCq method. Three groups of roots were sampled. Soybean SKIP16 (Glyma.12G051100) served as reference genes. Primers used are detailed in Supplementary Table S1 , and each data point was replicated three times.

Detection of POD activity and lignin content determination were performed on a Multiskan GO (Thermo Scientific, Waltham, Massachusetts, USA) according to the manufacturer’s instructions with a Peroxidase Activity Assay Kit (Boxbio, Beijing, China) and a Lignin content detection kit, micromethod (Solarbio, Beijing, China). For each plant sample, a triplicate procedure was executed to ascertain the precision and uniformity of the experimental outcomes.

The GmPOD53L coding region was amplified using a 2×Phanta^® Max Master Mix (Vazyme, Nanjing, China) and ligated into the PRI101-GFP using a ClonExpress^® II One Step Cloning Kit (Vazyme, Nanjing, China). The constructs (OE) were transformed into Agrobacterium rhizogenes K599 Chemically Competent Cell (Protein Interaction, Wuhan, China), with the PRI101-GFP empty plasmid serving as a control (EV₁). The 5-day-old soybean seedlings were obliquely cut off near the hypocotyl, a drop of bacterial mass was applied to the incision site and the seedlings were transplanted on wet vermiculite (Yang et al., 2021). Fluorescent hairy roots can be grown after 30 days of culture. The positive hairy roots were checked by a LUYOR-3415RG Hand-held Lamp (Luyor, Shanghai, China). In addition, qRT-PCR was used to detected the expression of target gene. The primers used were listed in Supplementary Table S1 .

Virus-induced gene silencing was performed using pTRV to silence target genes in soybean roots. A 300-bp fragment of the 5’ cDNA of GmPOD53L was ligated into the pTRV2 vector. The constructed vectors were transferred into Agrobacterium EHA105 Chemically Competent Cell (Protein Interaction, Wuhan, China). Agrobacterium-mediated transformations were carried out as described previously (Chen et al., 2024). A 1:1 (v/v) mixture of pTRV1with pTRV2 (EV₂) or pTRV2-GmPOD53L (KO) was injected around the roots of 4-day-old soybean plants at a rate of 5 mL per plant using a syringe. The process was repeated every 3 dpi, for a total of six applications. Samples were collected for gene expression analysis 4 days after the final inoculation. The primers used were listed in Supplementary Table S1 .

The suspensions of the second stage juveniles (J2s) were concentrated to 400pcs/ml through a 23μm sieve and then mixed 1:1 with a sterilized 0.2% water-agar to 200 pcs/mL (The final concentration of water-agar working solution is 0.1%). 5ml water-agar working solution was added into a 15ml centrifuge tube, and the roots of the plants were inserted into the bottom of the tube, so that the roots of the plants were completely immersed in the water-agar working solution (Each plant was inoculated with 1000 J2s). The outer wall of the centrifuge tube was wrapped with aluminum foil to ensure that the roots were protected from light and cultured in this state for two days. After two days, the plants were taken out, the water-agar working solution remaining in the roots was gently washed by running water, and then transferred to the sterilized vermiculite for further growth for 10 days.

Under suitable conditions, SCNs can develop from J2 to adult in about 14 days at the earliest. If the culture time is too long, some cysts formed by female adult will fall off into the soil, increasing the statistical error. In this study, after 12 days post-inoculation (dpi), the roots were stained using the acid fuchsin method (Bybd et al, 1983). The total number of SCNs and the number of different development stages were counted (Cook et al, 2012).

Graphpad Prism 9.0 and Microsoft Excel 2019 were used for data statistics and graph analysis. Comparisons between two groups were conducted using the Student’s t-test. Significance levels are denoted as follows: *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. Comparisons among multiple groups were performed using two-way ANOVA, with P < 0.01 considered significant. All values are presented as means ± standard deviation (SD) from at least three biological replicates.

Our previous research has found that CPT significantly reduces the number of nematodes in soybean roots (Kang et al., 2018; Xing et al., 2020). Here, we further investigated the effects of CPT pre-treatment on the growth and development of soybean plants, as well as its impact on SCN resistance in the roots systems. We first observations of the growth of soybean plants treated with CPT coating after reaching the two-leaf stage (7 day-old) revealed that CPT pre-treatment resulted in better growth compared to the control group ( Figure 1A ). Specifically, at 15 days post-inoculation (dpi), statistically significant differences (p < 0.05) were observed in the fresh ( Figure 2A ) and dry weights ( Figure 2B ) of the above-ground parts. However, no significant changes were detected in the fresh ( Figure 2A ) and dry weights ( Figure 2B ) of the below-ground parts.

The SCN life cycle involves hatching as J2 larvae, host penetration, syncytium formation, development, mating, and egg-laying. Soybean resistance to SCN mainly lies in impeding nematode infection, root development, and reproduction, thereby disrupting its normal life cycle. To further investigated the proportion of J2 of SCN in the root systems after CPT pretreatment. The plants were inoculated at the two-leaf stage, and the assessment was conducted 14 days post-inoculation (dpi). The results indicated that the proportion of J2 SCN in the roots was higher in all CPT-treated groups compared to the control group. Notably, the highest proportion of J2 was observed in plants treated with a CPT concentration of 1.2 mmol·L^-1, suggesting that this concentration effectively arrested the development of SCN at the J2 stage ( Figure 1B ). The results indicate that CPT treatment effectively enhances the resistance of soybean roots to SCN and promotes the growth of the above-ground parts of soybean plants.

Under abiotic stress, plants produce more tightly bound lignin, which strengthens cell walls and supports normal cellular functions. From the perspective of physical defense, we measured the lignin content in soybean roots under CPT treatment after SCN infection. The lignin content in soybean roots increased significantly after SCN infection, with a notable rise at 4–5 dpi, coinciding with the formation of syncytia by the H. Glycines. This indicates that under SCN stress, the lignin synthesis pathway in soybean roots is activated to resist the stress. In contrast, the CPT-coated treatment group maintained a higher lignin content from 1 dpi onwards. CPT significantly enhances the overall resistance of soybean roots. When SCN infected plants pre-treated with CPT, lignin levels rose significantly as early as 3 dpi and remained high thereafter ( Figure 3A ). This suggests that CPT treatment can induce soybean plants to produce more lignin in the early stages of SCN infection, thereby enhancing the plant’s physical defenses against nematode invasion.

POD regulates lignin monomer polymerization downstream in the lignin synthesis pathway and modulates ROS levels by adjusting H₂O₂ production and scavenging under stress. We further measured the changes in POD activity in soybean roots under treatments of SCN infection, CPT coating, and SCN infection after CPT coating pretreatment. The POD activity in soybean roots increased after SCN infection compared to the control group, indicating that the roots’ POD activity is activated in response to SCN stress. There was no significant difference in POD activity between the CPT-coated treatment and the control group. However, in the combined CPT coating pretreatment and SCN infection, POD activity only showed a statistically significant difference at 10 dpi ( Figure 3B ). These results suggest that CPT coating pretreatment delays the induction of POD activity under SCN stress, indicating that POD can actively respond to SCN. Additionally, the enhanced resistance of soybean plants after CPT coating means that lower POD activity is sufficient to defend against nematodes, thereby reducing damage caused by oxidative stress.

These findings presented above indicate that CPT (a specific treatment or compound, as contextually relevant) has a significant regulatory effect on both the lignin content and POD activity within the soybean root system. This dual-action mechanism of CPT not only enhances the structural integrity of the soybean roots thereby providing a robust defense mechanism against these harmful pathogens.

Previous research through combined transcriptomic and metabolomic analyses confirmed that CPT treatment enhances the activity of POD and the expression of several POD family genes (Kang et al., 2018). We further investigated the role of Class III POD gene, GmPOD53L, in regulating soybean resistance to SCN. qPCR-PCR revealed that GmPOD53L is significantly upregulated after SCN infection, with the highest expression level observed at 5 dpi, indicating that GmPOD53L can respond to SCN stress ( Figure 4 ).

To further investigated the function of GmPOD53L in regulation soybean roots resistance to SCN. The overexpression of GmPOD53L was achieved by Agrobacterium rhizogenes inducing hairy roots. qPCR was employed to detect the expression level of GmPOD53L in hairy roots. In the overexpression (OE) groups, the relative expression level of GmPOD53L was significantly higher than that of the empty vector (EV₁) control, ranging from 3- to 7-fold ( Figure 5A ). Moreover, laser detection of GFP fluorescence confirmed the successful transformation of the cultured soybean hairy roots, as evidenced by the distinct presence of positive GFP signals ( Figure 5B ). The transformed hairy roots were inoculated with SCN, and at 12 dpi, the roots were stained using the acid fuchsin method. A significant statistical difference was observed between the OE groups and the EV₁ control in terms of the total number of nematodes. In the OE groups, the total number of SCNs was lower than that in the EV₁ control ( Figure 5C ). When comparing the number of SCNs at the same developmental stage between the OE groups and the EV₁ control, significant statistical differences were found in the number of J2s, J4s, and adults. Specifically, the number of J2s was significantly higher in the OE groups, while the proportions of J4s and adults were significantly lower ( Figure 5D ).

To determine whether overexpression of GmPOD53L would lead to alterations in downstream products and affect resistance of soybean roots to SCN, lignin content and POD activity were measured. At 5 dpi with SCN, both lignin content ( Figure 5E ) and POD activity ( Figure 5F ) were found to be elevated in the overexpression (OE) groups compared to the empty vector (EV₁) control. These results demonstrate that overexpression of GmPOD53L can significantly enhance the resistance of soybean roots to SCN invasion, thereby retarding SCN development and conferring additional resistance to SCN.

The silencing of GmPOD53L was accomplished through Tobacco Rattle Virus (TRV)-mediated Virus-Induced Gene Silencing (VIGS). Compared with the normally growing plants, those subjected to VIGS exhibited significantly delayed development, characterized by varying degrees of mosaic patterns, chlorosis, and underdeveloped root systems ( Figure 6A ). The silencing of GmPOD53L was detected by qPCR. In pTRV1/pTRV2: GmPOD53L groups (KO), the relative expression level of the GmPOD53L gene was significantly downregulated, with a reduction of more than 50% compared to the EV₂ ( Figure 6B ).

The KO mutant lines were inoculated with SCN, and at 12 dpi, the roots were stained using the acid fuchsin method. Subsequently, the total number of SCNs and the number of nematodes at different developmental stages were counted. A significant statistical difference was observed between the KO groups and the EV₂ control group in terms of the total number of nematodes. Specifically, the total number of SCNs was higher in the KO groups than in the EV₂ groups ( Figure 6C ). Notably, the proportion of J2s was lower in the KO groups, whereas the proportions of J4s and adults were significantly higher than in the EV₂ control group ( Figure 6D ). Meanwhile, the POD activity and lignin content were measured in the KO lines. In the KO groups, both lignin content ( Figure 6E ) and POD activity ( Figure 6F ) and were found to be lower than in the EV₂ control. These results collectively suggest that silencing of GmPOD53L compromises soybean’s resistance to SCN, thereby facilitating increased SCN invasion.