The NbCBP1-NbSAMS1 Module Promotes Ethylene Accumulation to Enhance Nicotiana benthamiana Resistance to Phytophthora parasitica Under High Potassium Status

Abstract

Potassium (K) fertilization is crucial for plant resistance to pathogens, but the underlying mechanisms remain unclear. Here, we investigate the molecular mechanism by which the addition of K promotes resistance in Nicotiana benthamiana to Phytophthora parasitica. We found that N. benthamiana with high K content (HK, 52.3 g/kg) produced more ethylene in response to P. parasitica infection, compared to N. benthamiana with low-K content (LK, 22.4 g/kg). An exogenous ethylene application effectively increased resistance in LK N. benthamiana to the level under HK status, demonstrating the involvement of ethylene in the HK-associated resistance in N. benthamiana. Further, transcriptome analysis showed that NbSAMS1, encoding ethylene biosynthesis, was induced to upregulate P. parasitica about five times higher in HK than in LK N. benthamiana. NbSAMS1 overexpression enhanced resistance in LK plants, whereas NbSAMS1 silencing reduced resistance in HK plants, confirming its importance in conferring resistance. Furthermore, we identified a calcium-binding protein, NbCBP1, which interacted with NbSAMS1, promoting its expression in HK N. benthamiana. Silencing NbCBP1 compromised resistance in HK N. benthamiana, whereas its overexpression improved resistance in LK N. benthamiana. Notably, NbCBP1 protected NbSAMS1 from degradation by the 26S proteasome, thereby sustaining ethylene accumulation in HK N. benthamiana in response to P. parasitica infection. Thus, our research elucidated some mechanisms of the NbCBP1-NbSAMS1 module associated with disease resistance in HK N. benthamiana.

1. Introduction

Potassium (K) is a critical macronutrient for plant growth and development, and is essential for various physiological and biochemical processes, such as protein synthesis, osmoregulation, ionic balance, and enzyme activation. K also emerges as a positive regulator of plant defense. Numerous studies have demonstrated the protective benefits of K fertilizer application against diverse plant pathogens [1]. Sufficient K or high K (HK) levels have been associated with enhanced resistance to pathogens, while insufficient K can compromise plant immunity [2]. Optimal potassium levels vary across different plants. For N. benthamiana, the optimum foliar potassium content is 4.0%, which is the lower limit of the optimal potassium level for tobacco [3]. Improper potassium fertilization can affect various aspects of plant health, soil quality, and the environment. In areas where the average K content of foliage is well below optimum [4,5,6], these deficiencies have contributed to an increased incidence of apple Valsa canker. However, applications of potassium causing tissue to exceed recommended levels may lead to significant decreases in yields, and may even result in nutrient loss due to excessive leaching [7]. In field settings, the application of K fertilizer has been shown to reduce the impact of Cytospora mali damage on apples [8]. The plant hormone, ethylene, is a key regulator in various aspects of plant processes, especially as a defense response to pathogen infection [9]. Exogenous ethephon could protect plants against the necrotrophic fungus Botrytis cinerea [10]. Ethylene accumulation has also been reported to enhance plant resilience to various abiotic stresses [11,12]. Thus, an understanding of the relationship between K supply and plant resistance can help us design agricultural strategies that support crop nutrition and enhanced resilience [13].

The S-adenosyl-l-methionine synthetase gene (SAMS) plays a pivotal role in ethylene biosynthesis. SAMS also contributes to plant development and stress responses [14,15]. For example, CLCuMuV C4 suppresses transcriptional and post-transcriptional gene silencing-based antiviral defense in cotton by targeting and inhibits NbSAMS2 [14]. PlAvh202 promotes the degradation of LcSAMS through 26S proteasome-mediated pathways, reducing LcSAMS-catalyzed ethylene production and weakening plant resistance to Peronophythora litchi [16]. Thus, SAMS catalyzed ethylene synthesis may be a potential key process in plant resistance to pathogens.

Plants have evolved multi-level defense mechanisms to combat pathogen infections [17]. Calcium signaling serves as a secondary messenger in signal transduction, regulating physiological and biochemical processes in plants and improving plant resistance [18]. Plant calcium-binding proteins include calmodulin (CaM), calmodulin-like (CML), calcium-dependent protein kinase (CDPK), and calcineurin B-like proteins (CBL) [19]. These proteins recognize calcium signals and trigger defense responses, such as defense genes activations, ROS burst, and the production of nitric oxide, and plant hormones such as salicylic acid, jasmonic acid, and ethylene [20]. Numerous studies have found that potassium nutrition is closely related to the expression of CML genes [21,22]. The increase in intracellular calcium ion concentration coincides with rapid membrane depolarization and potassium efflux during the initial phases of pathogen response [23]. Thus, these results imply that there may be a close relationship between calcium-binding proteins and potassium in activation of plant disease.

In this study, we found that ethylene accumulation plays an essential role in the resistance of N. benthamiana to P. parasitica. Notably, we found that a calcium signaling pathway protein, NbCBP1, interacts with and stabilizes NbSAMS1 by blocking 26S proteasome ubiquitination/degradation, thereby sustaining ethylene accumulation under HK and during pathogen infection. Overall, we found that the NbCBP1-NbSAMS1 module promoted ethylene accumulation and was responsible for enhanced resistance in HK N. benthamiana.

2. Results

2.1. High Potassium Levels Promote Ethylene Accumulation After Inoculation

We grew N. benthamiana under low potassium and high potassium conditions for three weeks to obtain plants with low potassium (LK) and high potassium (HK) contents (Table S1). The leaf K assessment showed that the K content in HK N. benthamiana was 52.3 g/kg, which was significantly higher than that of LK (22.6 g/kg) (Table S1). And there were no significant differences in the levels of nitrogen (N) and phosphorus (P) among all HK and LK plants (Table S1). Typically, this high planta K content was the optimal or standard level for both resistance responses and the primary growth of N. benthamiana [24]. Further transcriptomic analysis revealed that the expressions of genes involved in ethylene (ET) synthesis and signaling were more active in HK N. benthamiana infected by P. parasitica (IHK), than in infected LK N. benthamiana (ILK) (Figure 1a). Among 158 ET-related genes, the expression levels of 120 were higher in IHK N. benthamiana than those in ILK (Figure 1a), indicating the involvement of ethylene-related processes in the high resistance of HK N. benthamiana to P. parasitica.

Ethylene production was measured in N. benthamiana leaves on a fresh-weight basis. No significant differences were observed in ethylene levels between non-inoculated LK and HK plants (Figure 1b). However, upon P. parasitica inoculation, a significant increase in ethylene production was detected in IHK plants (55.52 μL·kg⁻¹·h⁻¹) compared to ILK plants (17.25 μL·kg⁻¹·h⁻¹) (Figure 1b). This indicated that HK plants produced higher levels of ethylene in response to pathogen infection.

To further assess the role of ethylene in plant disease resistance, the effect of exogenous ethephon (ETH) was explored. At 36 h post-inoculation (hpi), high potassium plants treated with ethephon (HK-ETH) 24 h earlier, exhibited no lesions on leaves, similar to low potassium plants with ethephon treatment (LK-ETH), which showed reduced lesions diameter (0.4 ± 0.2 cm). High potassium plants without ethephon treatment (HK-MOCK) developed small lesions (0.8 ± 0.2 cm) on leaves, while low potassium plants without ethylene treatment (LK-MOCK) showed significantly larger lesions (4.1 ± 0.2 cm) (Figure 1c,d). The results revealed that ethylene-treated plants exhibited high resistance to P. parasitica, even in LK N. benthamiana, demonstrating that ethylene-enhanced N. benthamiana resistance to P. parasitica was related to K status.

2.2. NbSAMS1 Positively Regulates Nicotiana benthamiana Resistance to Phytophthora parasitica

SAMS1 plays a pivotal role in ethylene biosynthesis, which is critical for plant defense. Previous research has also demonstrated its role in plant development and stress responses [14,15]. To understand the relationships between K contents in plants and ethylene synthesis, the expression of NbSAMS1 in HK and LK N. benthamiana was analyzed. No significant differences in NbSAMS1 expression were observed between non-inoclated HK and LK plants. However, P. parasitica inoculation significantly upregulated NbSAMS1 expression in HK N. benthamiana, which was about five times higher than that in LK (Figure 2a). This indicated that pathogens could enhance the expression of NbSAMS1, especially in plants with HK status. We also found that the expression level of pathogenesis-related (PR) genes NbPR1, NbPR4, NbPR2, and NbPAL were significantly upregulated compared to the control (Figure 2b), implying that NbSAMS1 may act as a positive regulator of plant immunity.

To investigate the role of NbSAMS1 in plant resistance, we constructed overexpression (OENbSAMS1) and silencing (VNbSAMS1) vectors and generated transgenic plants. RT-qPCR confirmed increased overexpression of NbSAMS1 in both HK and LK N. benthamiana (Figure S1a); however, NbSAMS1 expression was reduced to 60% in silenced N. benthamiana (Figure S1b). A pathogenicity assay revealed that NbSAMS1 overexpression significantly reduced the lesion size in N. benthamiana even with LK status, which was smaller than that of the non-overexpressed IHK control (Figure 2c). The lesion diameter after NbSAMS1 silencing was significantly enlarged in N. benthamiana with HK status (Figure 2d). These results showed that NbSAMS1 was a positive regulator of N. benthamiana resistance to P. parasitica.

2.3. NbSAMS1 Interacts Directly with NbCBP1

We constructed a hub network of NbSAMS1 and NbCBP1 expression along with their related genes to better understand their functional roles in plant defense. The analysis showed a strong correlation between NbCBP1 and NbSAMS1, suggesting their interconnected roles in plant resistance. Additionally, these genes were associated with the expression of other genes involved in key defense mechanisms, such as transcriptional regulation, calcium signaling transduction, and kinase activity, further underscoring the importance of NbCBP1 and NbSAMS1 in plant defense. The predicted network showed that NbCBP1 might act as an upstream regulatory protein of NbSAMS1 (Figure 3a). This was further supported by molecular docking analysis, which indicated a strong interaction between the two proteins, with a binding energy of ΔiG = −16.1 kcal/mol (Figure 3b, Table S4). To confirm this interaction, we used a yeast two-hybrid (Y2H) assay (Figure 3c). The results indicated direct interaction between NbCBP1 and NbSAMS1. Then, the interaction between NbCBP1 and NbSAMS1 was confirmed by CoIP in vivo (Figure 3d).

2.4. NbCBP1 Affects Ethylene Accumulation and Positively Regulates Nicotiana benthamiana Resistance

To investigate whether NbCBP1 is related to K levels, the expression of NbCBP1 was assessed in HK and LK N. benthamiana. The results showed that NbCBP1 was significantly induced in HK and LK N. benthamiana following P. parasitica challenge. The expression of NbCBP1 was much higher, about six times in IHK than in ILK N. benthamiana. However, there were no differences in NBCBP1 expression between HK and LK N. benthamiana. These results showed that NbCBP1 could be differentially induced by pathogen infection and that K content could promote its upregulation (Figure 4a). To assess the impact of NbCBP1 on plant resistance, we analyzed the transcription levels of PR genes, and found that the expression of NbMYC2, NbPAL, and NbPR4 was significantly upregulated (Figure 4b), implying that NbCBP1 may act as a positive regulator of plant immunity.

To assess whether NbCBP1 affects plant disease resistance, we overexpressed NbCBP1 in N. benthamiana. RT-qPCR analysis confirmed NbCBP1 overexpression in both HK and LK N. benthamiana (Figure S2a). Then, we inoculated the leaves with P. parasitica, and at 3 dpi, we found that the lesions on LK or HK N. benthamiana overexpressing NbCBP1 were significantly smaller than those of the corresponding non-overexpressed control, indicating that NbCBP1 could enhance host immunity (Figure 4c). We further investigate the function of NbCBP1 in N. benthamiana using the VIGS system. After infiltration, the efficiency of gene silencing was verified by RT-qPCR. The results showed that the expression levels of NbCBP1 in both LK and HK N. benthamiana were significantly suppressed (Figure S2b). Pathogenicity assays also showed that the lesion diameters on HK N. benthamiana leaves with NbCBP1 silencing were larger than that in HK controls (Figure 4d). In parallel, NbCBP1 silencing did not affect lesion development in LK leaves (Figure S2c). These results supported the positive regulatory effect of NbCBP1 on plant disease resistance.

Next, we investigated the role of NbCBP1 in ethylene production in N. benthamiana. Ethylene accumulation was significantly higher in the NbCBP1-overexpressing IHK plants, which produced 81.8 μL·kg⁻¹·h⁻¹, compared to the NbCBP1-overexpressing ILK plants, which had 58.9 μL·kg⁻¹·h⁻¹. Ethylene production was considerably lower in the NbCBP1-silenced plants, with the VNbCBP1-IHK plants showing 7.2 μL·kg⁻¹·h⁻¹ and the VNbCBP1-ILK plants showing 2.9 μL·kg⁻¹·h⁻¹ (Figure 4e). These silenced plants had significantly lower ethylene production, but they were not different from each other (Figure 4e). Additionally, we also observed that silencing NbCBP1 affected plant height, with LK N. benthamiana showing a height of 7.1 cm compared to 11.5 cm in HK N. benthamiana (Figure 4f). These findings indicated that NbCBP1 played a crucial role in ethylene synthesis and could be upregulated by pathogen infection, especially in HK plants.

2.5. NbCBP1 Blocks Ubiquitination/Degradation to NbSAMS1 to Promote Ethylene Accumulation in HK N. benthamiana

The predicted interaction network showed that CBP1 might be an upstream regulatory protein of SAMS1 (Figure 3a). We then overexpressed NbCBP1 and quantitatively measured the expression of NbSAMS1, and found no significant difference in NbSAMS1 expression in N. benthamiana among LK, HK, and ILK plants. However, NbSAMS1 expression significantly increased in IHK plants (Figure 5a). After silencing NbCBP1, there were no differences in NbSAMS1 expression in IHK plants compared with LK, HK, and ILK plants (Figure 5b). These results suggested that NbCBP1 is an upstream regulation protein of NbSAMS1, and the high expression of NbSAMS1 in IHK N. benthamiana was induced by NbCBP1.

Previous studies have shown that SAMS1 undergoes protein degradation influenced by ubiquitination in several plants. For example, OsFBK12 (a subunit of E3 ligase) targets OsSAMS1 for degradation in rice [25]. To understand whether NbSAMS1 is affected by ubiquitination, we used AlphaFold3 to predict the potential interactions among NbCBP1, the E3 ligase, and NbSAMS1. The results showed that there may be a direct interaction between NbCBP1 and NbSAMS1, as well as between NbSAMS1 and E3 ligase (Figure S3a,b), thus implying that the E3 ligase may mediate the degradation of SAMS1. The prediction of the interaction among these three proteins suggested that NbCBP1 exhibits a strong binding affinity for SAMS1 (Figure S3c). This suggests a competitive relationship between CBP1 and the E3 ligase for binding to NbSAMS1. Thus, we hypothesize that NbCBP1 may prevent the ubiquitination of E3 ligase and subsequent degradation of NbSAMS1.

To test the above hypothesis, we performed a Western blot to compare the molecular mass patterns of NbSAMS1. The result demonstrated that bands with higher molecular mass were found in the HK N. benthamiana leaves with NbSAMS1 overexpression; however, lower molecular mass bands were found in LK leaves (Figure 5c). To understand the differences caused by 26S proteasome ubiquitination, LK N. benthamiana leaves expressing NbSAMS1 were treated with 26S proteasome inhibitor MG132. We found that NbSAMS1 was stable in the presence of MG132 in LK status (Figure 5d), whereas in the DMSO (control), NbSAMS1 showed a gradual decrease. These findings suggested that NbCBP1 might protect NbSAMS1 from ubiquitination/degradation by the 26S proteasome under HK status.

3. Discussion

Ethylene is one of the six major plant hormones, which are important regulators in plant growth, development, and reproduction. Ethylene has also been found to play an important role in plant disease resistance. The synthesis of ethylene is affected by a variety of mineral elements, such as N, P, Ca, Fe, Mn, Cu, S, Co, B, Mg, and Cd [26,27], and by pathogens. Ca availability has been shown to enhance plant disease immunity by promoting ethylene production. In mung beans, increased Ca significantly boosted ethylene production [26,28]. Many studies have shown that ethylene production is induced by infection with pathogens. For instance, ethylene is induced in tobacco plants infected with Phytophthora parasitica [29], in rice infected with Magnaporthe oryzae [30], and in carrots infected with Botrytis cinerea [31]. PTI and ETI utilize the PICI1-OsMETS-ethylene cascade, and ETI-PTI integration enhances rice broad-spectrum blast resistance [32]. SAMS1 is a key gene in ethylene biosynthesis and plays a crucial role in plant disease resistance, as its expression is induced by various biotic stresses, conferring increased tolerance [33,34]. In litchi, SAMS1 positively regulates ethylene biosynthesis and plant immunity against Peronophythora litchii [16]. Silencing the SAMS enhances plant susceptibility to CLCuMuV infection in N. benthamiana [14]. These studies show that ethylene plays a significant role as a modulator of plant disease resistance. Consistent with these findings, our results showed that both the high expressions of the NbSAMS1 and the increased ethylene production all contribute to the resistance enhancement of N. benthamiana to P. parasitica. Previous studies showed that potassium deprivation induces ethylene production [35], and that adequate potassium nutrition suppresses ethylene evolution [36]. Our results showed that for ethylene evolution in N. benthamiana, there were no differences between high-potassium and low-potassium conditions. Unexpectedly, we found that the P. parasitica infection significantly promoted ethylene accumulation in HK N. benthamiana, which was about three times higher than that in LK N. benthamiana. Our findings demonstrated that high potassium or adequate potassium status could promote ethylene synthesis and improve plant resistance under biotic stress.

PR genes are involved in plant defense by activating SAR and ISR via the salicylic acid (SA), jasmonic acid (JA) or ethylene signaling pathways [37]. Additionally, NbPAL, involved in the phenylpropanoid pathway, promotes the production of phenolic compounds such as lignins, which reinforce physical barriers and boost immune responses against pathogens [38]. Our findings showed that NbPR1, NbPR2, NbPR4, and NbPAL were upregulated in NbSAMS1 overexpressed plants, indicating the pivotal roles of PR-related genes in enhancing N. benthamiana resistance, and supporting their involvement in plant immunity pathways.

The calcium signaling pathway significantly affects plant immunity, and various calcium-binding proteins have been shown to be influenced by potassium nutrition. For example, potassium nutrition can affect calcium signaling, e.g., the calcium sensor protein genes OsCML1, OsCML18, OsCML20, and OsCML31 are up-regulated in rice during low-K stress [39]. The Ca²⁺-CaM signaling pathway promotes K absorption under K stress [40]. The calcium signaling was reported to be related to ethylene signaling. Following infection, TaCML36 expression increases in the ethylene signaling pathway, resulting in increased resistance to sharp eyespot [41]. Our study identified the novel CML family protein NbCBP1 and its expressions in N. benthamiana were differentially induced in plants with varying potassium status by pathogen infection. High potassium N. benthamiana with pathogen challenge can promote ethylene accumulation and resistance to P. parasitica. These findings suggested that potassium affected plant immunity via calcium and ethylene signaling pathways. We also found that overexpression could enhance plant resistance even in LK plants, implying that CBP1 could be potential resistance gene for developing transgenic plants. To our knowledge, this is the first report on CBP1 involvement in promoting ethylene accumulation and plant immunity.

As a major macro-nutrient, potassium content profoundly affects disease severity [13]. Earlier research has shown that sufficient potassium supply reduces lipid peroxidation and preserves lipid homeostasis, thereby mitigating the adverse effects of sheath rot caused by Sarocladium oryzae on rice [42]. Sufficient potassium supply (0.93%) in apple branches enhances resistance to Cytospora mali through the promotion of several antifungal secondary metabolites, especially sufficient coumarin accumulation [43]. Apart from the accumulation of reactive oxygen species and antifungal metabolites, the nucleotide-binding domain and leucine-rich repeat resistance genes (NLR) are activated in high-potassium N. benthamiana [24]. These research findings show that the mechanisms of K-enhanced plant immunity must be highly complicated. This study found that high potassium status enhanced plant immunity by promoting ethylene accumulation. Furthermore, the key calcium-binding protein NbCBP1 was identified. On the one hand, overexpression of NbCBP1 in N. benthamiana promoted the high expression of NbSAMS1, and silencing NbCBP1 in N. benthamiana led to a lower expression of NbSAMS1, implying that NbCBP1 caused high expression of NbSAMS1 in HK plants upon pathogen infection. On the other hand, we found that NbCBP1 interacted with NbSAMS1 and protected it from ubiquitination/degradation in HK plants, which was particularly important after pathogen infection. Taken together, these results supported the hypothesis that upon pathogen infection, NbCBP1 was induced and highly expressed in high potassium plants, which promoted high NbSAMS1 expression. NbCBP1 further interacted with NbSAMS1, protecting it from ubiquitination degradation, keeping NbSAMS1 at a high level, and enhancing ethylene accumulation and plant disease resistance (Figure 6). Our results support the role of NbCBP1 in stabilizing NbSAMS1 via potential E3 ligase ubiquitination. Future research will focus on identifying the details of ubiquitination proteins involved in the ubiquitination of NbSAMS1. Several observations indicate that applying ethylene before infection with pathogens reduces the disease severity [44]. Our results showed that exogenous ethephon treatment on LK plants significantly increased plant disease resistance, suggesting ethylene functions in increasing plant immunity. The exogenous ethylene application activates resistance responses against a broad spectrum of pathogens. This could be used as an additional strategy to protect plants from diseases in low potassium fields.

In conclusion, we uncovered a novel mechanism by which K influences ethylene accumulation and plant immunity. This mechanism is critical for enhancing plant pathogen resistance and highlights a significant dimension of the role of potassium in plant defense.

4. Materials and Methods

4.1. Plant Materials

N. benthamiana seedlings were transplanted into plastic pots with sterile mixtures of vermiculite and soil substrate at 1:1 (v/v) [45]. Subsequently, the seedlings were grown in a modified Hoagland’s nutrient solution (Table S2). To achieve high-K plants (4% to 6%) [46], the solution contained two-fold the amount of KNO₃ (0.758 g/L). Conversely, to obtain low-K plants (<2%), the solution had no KNO₃ (0 g/L). To ensure an equal nitrogen level in both solutions, 0.3 g/L NH₄NO₃ was added into the 0 K nutrient solution. After three weeks of incubation in the growth chamber at 23 °C with a 16 h light and 8 h dark photo period and ~60% humidity, the N, P, and K content in the dry leaf weight were measured, and the HK and LK N. benthamiana leaves were harvested for the further experiment. There were three replicates for each treatment, and the entire experiment had three repetitions.

4.2. Pathogen Inoculation and Disease Severity

The Phytophthora parasitica var. nicotianae strain was grown on PDA at 25 °C. The strain was provided by Prof. Xili Liu (Northwest A&F University), and it was used in previous research to infect N. benthamiana [43]. Hyphal plugs with a 55 mm diameter of P. parasitica were inoculated into nonwounded detached leaves from N. benthamiana seedlings. At 3 dpi, the inoculum plugs were removed, and representative leaves were photographed for evaluation. Each inoculation experiment had at least three replicates, and the experiment was conducted three times.

4.3. Construction of Vectors and Genetic Transformation in N. benthamiana

TRV vectors were constructed by cloning 350 bp DNA segments of NbSAMS1 (Ni-ben101Scf04643g02010.1) and its target gene NbCBP1 (Niben101Scf08035g00009) of N. benthamiana using a primer pair containing EcoRI and BamHI sites and then ligating them to pTRV2 (Table S3). Appropriate corresponding recombinant vectors were introduced into Agrobacterium GV3101, which was then used to transform both LK and HK N. benthamiana as previously described [47]. The control group consisted of an Agrobacterium solution with an empty vector. Silenced N. benthamiana leaves were subsequently inoculated with P. parasitica as above. RT-qPCR was used to analyze NbCBP1 and NbSAMS1 transcript levels in plants at 3 days post-incubation. The full-length cDNA of NbCBP1 (Niben101Scf08035g00009) was amplified using a primer pair containing BamHI sites and then cloned into the pBIN-eGFP overexpression vector, and full-length cDNA of NbSAMS1 (Niben101Scf04643g02010.1) was amplified using a primer pair containing BamHI sites and then cloned into the MYC overexpression vector (Table S3). The overexpressing N. benthamiana was inoculated with P. parasitica and analyzed following agro-infiltration using RT-qPCR, as previously described in [47]. There were three replicates of each treatment, and the entire experiment had three repetitions.

4.4. Total RNA Extraction and RT-qPCR Analysis

The Omega Plant RNA Kit (Omega Bio-Tek) was used to extract the total RNA from N. benthamiana leaves and then Easy Script cDNA Synthesis Kit (Transgene, Beijing, China) was then used to reverse-transcribe the whole RNA to cDNA. Gene-specific primers and SYBR Green staining were used to measure the levels of gene expression in triplicate. For RNA samples, the expression of NbEF1a served as an internal standard. We extracted and reverse-transcribed the RNA to cDNA from pathogen-inoculated N. benthamiana leaves with HK and LK status. Using these gene-specific primers, RT-qPCR was used to measure the gene expression with three biological replicates for each treatment, and three technical replicates per biological sample. Gene-specific primers were synthesized based on the cDNA sequences (Table S3). The 2^−△△CT technique was used to analyze the RT-qPCR data.

4.5. Protein Extraction and Western Blotting

Agroinfiltrated HK and LK N. benthamiana leaves were collected and ground in liquid nitrogen. Total proteins were extracted with RIPA extraction buffer (17). After adding the extracted and purified recombinant proteins to the loading buffer sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), they were boiled and centrifuged. Proteins were transferred to polyvinylidene difluoride membranes after the supernatants were obtained from different treatments, and then analyzed by PAGE. The membranes were incubated using the appropriate antibodies for the detection of NbSAMS1 and NbCBP1 protein [48].

4.6. IP-MS Analysis for Target Gene Identification

For IP-MS assays, the recombinant plasmid of NbSAMS1 was fused with the MYC tag to acquire the NbSAMS1-MYC protein. The MYC fused protein was overexpressed in N. benthamiana leaves. The supernatant (about 1 mL) of total proteins was extracted from both HK and LK N. benthamiana leaves and combined with magnetic beads that contained the GFP antibody at 4 °C overnight by a vertical mixer. The beads were then collected, washed six times with an IP buffer, and finally suspended beads in 120 μL IP buffer. The protein complex was assessed by immunoblotting using anti-MYC antibody to obtain candidate interactants.

For mass spectrometry analysis, the lyophilized protein samples were dissolved using 10 μL of solution and then centrifuged at 14,000× g at room temperature for 20 min to remove debris. The mobile phases of liquid chromatography consisted of Phases A that contained 100% water, 0.1% formic acid and Phase B contained 80% acetonitrile, 0.1% formic acid. A total of 1 μg of the supernatant was used for detection. The analysis was conducted using the EASY-LC™ 1200 UHPLC system (Thermo Fisher, Waltham, MA, USA) connected to a Q Exactive HF-X mass spectrometer (Thermo Fisher, Waltham, MA, USA) operating in the data-dependent acquisition (DDA) mode. A homemade C18 Nano-Trap column (2 cm × 75 μm, 3 μm) was used to trap the peptide from the sample. Using linear gradient elution, peptides were separated on a handmade analytical column (15 cm × 150 μm, 1.9 μm). Peptide were ionized using Nanospray FlexTM (Thermo Fisher, Waltham, MA, USA) (ESI) with spray voltage of 2.3 kV, and an ion transport capillary temperature was set at 320 °C to facilitate efficient ionization. The isolated peptides were examined using Q Exactive HF-X (Thermo Fisher, Waltham, MA, USA). The mass spectrometer was set to a resolution of 120,000 at m/z 200 for the full screen which covered the m/z from 350 to 1500, and an automated gain control (AGC) target was set of 3 × 106. The maximum ion injection period was 80 ms. The higher energy collisional dissociation (HCD) was used to select and fragment the 30 most abundant precursor ions from the entire scan. These were then subjected to MS/MS analysis with a resolution of 15,000 (at m/z 200), an automatic gain control (AGC) target value of 5 × 106, a maximum ion injection time of 100 ms, a normalized collision energy of 27%, an intensity threshold of 5 × 103, and a dynamic exclusion parameter of 20 s. The raw data obtained from the mass spectrometer were analyzed to identify potential interacting proteins.

4.7. Co-IP Assays

The Co-IP assay was conducted using the procedure outlined in [17]. The recombinant plasmid of NbCBP1 was fused with the GFP tag for acquiring NbCBP1-GFP proteins, while the NbSAMS1 were fused with the MYC tag for acquiring NbSAMS1-MYC proteins, respectively. GFP and MYC fusion proteins were co-expressed transiently in the leaves of N. benthamiana. The supernatant (approximately 1 mL) of total proteins was extracted from N. benthamiana leaves and combined with magnetic beads containing the GFP antibody using a vertical mixer at 4 °C overnight. The beads were collected, washed six times with an IP buffer, and then resuspended in 120 μL IP buffer. The protein complex was detected by immunoblotting using anti-GFP and anti-MYC to detect interaction. The experiment was repeated 3 times.

4.8. Y2H Assay

The Y2H assays were conducted using the ProQuest Two-Hybrid System (Invitrogen, Carlsbad, CA, USA). The protein-coding regions of each target were inserted in-frame into the pGADT7 vector to function as the bait. The open reading frames of the intracellular C-terminal segments of NbCBP1 were inserted into the pGBKT7 vector to be used as prey (Table S3). Positive clones were identified after the co-transformation of yeast (Y2H gold strain) and screening on SD/Leu-Trp-His plates. They were then grown on SD/Leu-Trp-His-Ade medium with varied concentrations of ABA and X-α-gal or assayed with β-galactosidase (X-gal). The experiment was conducted three times.

4.9. Exogenous Etephon Treatment

For the exogenous ethylene treatment, a solution was prepared by mixing 499 mL of distilled water, 1 mL of 40% ethephon (a plant growth regulator that releases ethylene), and 150 µL of organic silicon surfactant. The final concentration of ethephon in the solution was 0.08%. The prepared solution was thoroughly mixed and sprayed uniformly onto the leaves of N. benthamiana plants. The plants were left undisturbed for 24 h to allow the solution to be absorbed. The treatment conditions were kept consistent for the experimental groups, which included HK and LK N. benthamiana. Each treatment was repeated three times, and the entire experiment was conducted in triplicate.

4.10. Quantification of Ethylene

N. benthamiana leaves were treated with agroinfiltration, and after 24 h they were detached and weighed. Leaves were sealed in a 10 mL glass vial at 25 °C for 6 h. To quantify ethylene concentration, a 1 μL gas sample from the head space was injected into the gas chromatography (Agilent 7890B, Santa Clara, CA, USA) using a gas-tight syringe (Hamilton, Reno, NV, USA). The column (Agilent, GS-Alumina; 50 m × 530 μm × 0 μm) was maintained at 50 °C for 3 min. The temperatures for sample entrance and the hydrogen flame ionization detector (FID) were 200 °C and 300 °C, respectively. The peak area in the chromatogram was utilized to calculate ethylene concentration by comparison to the standard curve. Three biological replicates were included for each treatment.

4.11. In Vivo Protein Degradation and Western Blotting

NbSAMS1 protein degradation by the 26S proteasome was tested in vivo using A. tumefaciens-mediated transient expression in N. benthamiana leaves. At 48 hpa, 100 mM MG132 (Aladdin Industrial Corporation, Shanghai, China) or 0.5% (v/v) DMSO was infiltrated into N. benthamiana leaves, and total protein was extracted for Western blot at 0 h, 4 h, and 8 h.

4.12. Computational Prediction of Protein–Protein Interactions

First, protein sequences of NbCBP1, NbSAMS1, and E3 ligase were downloaded from the N. benthamiana genome database (https://solgenomics.net/organism/Nicotiana_benthamiana/genome (accessed on 16 June 2024)). These protein structure were predicted using AlphaFold3 (https://alphafold.ebi.ac.uk/ (accessed on 18 June 2024)) and protein–protein docking simulations were performed using default parameters. The binding interfaces between NbCBP1, NbSAMS1, and E3 ligase were examined, and binding affinity was calculated to evaluate the possibility of direct interactions using PDBePISA online server (https://www.ebi.ac.uk/msd-srv/prot_int/ (accessed on 24 June 2024)). Finally, the predicted complex models were visualized and analyzed using PyMOL (version 3.0) and Chimera software (version 1.17.1).

4.13. Statistical Analysis and Graphical Presentaion

All data were analyzed using one-way ANOVA and Student’s t-tests in Prism version 8 (GraphPad, San Diego, CA, USA). The data are presented as mean ± standard deviation (SD) and analyzed using Student’s t-test. Asterisks indicate statistical significance (** p < 0.01; * p < 0.05; ns = not significant), and different letters indicate significant differences (p < 0.05).