Screening and Validation of Interacting Proteins of Receptor-like Cytoplasmic Kinase OsRLCK118 Involved in Rice Blast Resistance

Abstract

Rice blast, caused by Magnaporthe oryzae, severely threatens global rice production. Although the receptor-like cytoplasmic kinase OsRLCK118 positively regulates rice immunity, its downstream signaling mechanism remains unknown. To systematically identify OsRLCK118-interacting proteins, we performed immunoprecipitation–mass spectrometry (IP-MS) and a yeast library screen, yielding 781 and 287 candidates, respectively, with 35 overlapping hits. Among these, OsSAMS1, a known positive regulator of blast resistance, was selected for validation. Membrane yeast two-hybrid, split-luciferase complementation, and co-immunoprecipitation assays confirmed the physical interaction between OsRLCK118 and OsSAMS1. Furthermore, in vitro kinase assays showed that OsRLCK118 specifically phosphorylates OsSAMS1. These results uncover a novel signaling axis connecting pathogen recognition to ethylene biosynthesis via OsRLCK118-dependent phosphorylation of OsSAMS1, providing both mechanistic insight into rice immunity and potential genetic targets for resistance breeding.

1. Introduction

Rice (Oryza sativa L.) is one of the most important food crops in the world. Rice blast, caused by Magnaporthe oryzae infection, is a worldwide disease that has shown a rising trend in recent years [1]. Rice blast not only causes a significant yield reduction or complete loss in severe cases but also affects rice quality [2,3]. Receptor-like kinases (RLKs) have extracellular receptor structural domains, transmembrane structural domains, and intracellular serine and threonine kinase structural domains for receiving external signals. Receptor-like cytoplasmic kinases (RLCKs) are defined by the absence of extracellular and transmembrane domains. They harbor a cytoplasmic kinase domain similar to that of RLKs [4]. RLCKs, as early hubs of plant immune signaling, not only regulate pathogen-associated molecular pattern (PAMP)/microbe-associated molecular pattern (MAMP)-triggered immunity (PTI) but also participate in disease-resistance responses, such as reactive oxygen species (ROS) bursts and mitogen-activated protein kinase (MAPK), and further affect rice development and abiotic stress responses. The systematic analysis of the functional network and mechanism of the OsRLCK family in rice will provide theoretical support for multi-dimensional disease-resistance breeding. This research holds significant value for both fundamental research and breeding applications.

Pattern-recognition receptors (PRRs), localized in the plasma membrane of plant cells, recognize extracellular pathogen-related molecular patterns that activate plant PTI. Further research shows that PTI and effector-triggered immunity (ETI) do not function independently, but there is a synergistic amplification [5]. A large number of studies have now shown that the signaling mediated by RLCKs downstream of many PRRs in response to PAMPs is critical for the development of quantitative resistance to bacterial and fungal pathogens. For example, in the immune response of Arabidopsis thaliana, RLCK BOTRYTIS-INDUCED KINASE1 (BIK1) plays a key role downstream of PRRs. When BIK1 is transphosphorylated by Elongation Factor Thermo Unstable Receptor (EFR) on two specific residues, S₈₉ and T₉₀, BIK1 is able to translocate to the nucleus and regulate the phosphorylation state of WRKY transcription factor, which in turn triggers an immune response [6]. In addition, BIK1, upon sensing PAMP, directly interacts with and phosphorylates Respiratory Burst Oxidase Homologue (RBOHD), which enhances plant immunity by ROS burst and stomatal closure [7,8]. Medium-chain 3-hydroxy fatty acids (mc-3-OH-FAs) from Gram-negative bacteria were found to be novel PAMPs in Arabidopsis. The Arabidopsis G-type lectin receptor kinase PRR LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE) recognizes 3-OH-C10,0 in mc-3-OH-FAs and undergoes tyrosine 600 phosphorylation, which in turn phosphorylates the BIK1-related protein PBS1-like 34/35/36 kinase that activates the immune response [9].

In rice, chitin induces OsCEBiP to bind to OsCERK1, and activated OsCERK1 phosphorylates the downstream component OsRLCK185, which then activates the immune response in rice [10]. To defend against pathogen infection, NLRs in plants recognize specific effector proteins and trigger ETI, including ROS bursts, upregulation of intracellular calcium ions, and MAPK cascade responses [11,12,13]. For example, rice OsCERK1 phosphorylates OsRLCK185, which further phosphorylates OsMAPKKKε, activating the MAPK cascade and the immune response [11]. In addition to regulating MAPK, RLCKs also phosphorylates RBOH, regulating ROS production [14]. OsRLCK176 and OsRLCK118 also function downstream of the monocot-specific receptor-like kinase SDS2 (SPL11 cell death suppressor 2). This then activates OsRbohB to activate ROS production and defense genes activation, ultimately orchestrating programmed cell death in response to chitin [15]. Rice genome-wide transcriptome analysis revealed that about 100 of the 379 OsRLCKs were differentially expressed at different times [16,17]. OsRLCK57/107/118/176 refers to four members of the rice OsRLCK VII subfamily involved in the regulation of ROS bursts induced by chitin and flg22 [18]. They also have an impact on rice growth and development, with OsRLCK57/107/118/176 negatively regulating the rice BR signaling pathway and affecting leaf tilt [19]. As such, RLCKs function as pivotal early signaling hubs in plant immunity [20]. Therefore, analyzing the signaling mechanism, specificity of biological functions, protein interactions, and structure–function studies of RLCK is imperative for understanding and improving resistance to diseases and abiotic stresses in rice. However, unlike well-studied members such as OsRLCK185 and BIK1, although it is known that OsRLCK118 positively regulates blast resistance [18,19], its specific downstream signaling mechanism remains a knowledge gap. Does it transduce signals by phosphorylating known MAPK cascade components or RBOH proteins [15]? Or does it involve novel, previously unidentified interaction targets and signaling pathways?

Therefore, to elucidate the unique downstream mechanisms of OsRLCK118, this study selected it as the research target, aiming to understand its functional specificity and refine the rice RLCKs-mediated immune regulatory network. We employed immunoprecipitation–mass spectrometry (IP-MS) and membrane yeast two-hybrid (MYTH) techniques to systematically screen for its direct interacting proteins. Our experiments elucidated the phosphorylation or protein–protein interaction relationships between OsRLCK118 and immune-related components at the molecular and cellular levels, thus enriching our understanding of the rice immune regulatory network. This will also provide a scientific basis for the in-depth exploration of blast resistance genes and the cultivation of new rice germplasms with disease resistance.

2. Materials and Methods

2.1. Experimental Materials

The wild-type material rice cultivar Zhonghua11 (ZH11), pSuper1300-GFP plasmid, pYBA1143-HA plasmid, pCAMBIA1300-NLuc, and pCAMBIA1300-CLuc were provided by the laboratory of Germplasm Resources Conservation and Utilization Center of the Rice Research Institute of Fujian Academy of Agricultural Sciences. The vector plasmids used in this experiment included yeast strain NMY51, pTSU2-APP positive control bait plasmid, pNuBG-Fe65 positive control prey plasmid, pPR3-N blank prey plasmid, pBT3-STE blank bait plasmid, His-pET-32a, plasmid His-pET-28 plasmid, and rice cDNA expression library, and they were provided by the laboratory of Prof. Dingzhong Tang, Plant Immunity Center, Fujian Agriculture and Forestry University [21].

2.2. Preparation and Transformation of Rice Protoplasts

Approximately 800 ZH11 seeds were evenly spread on cotton cheesecloth and incubated in the dark for 7–10 days for seedling formation [22]. Seeds and roots were removed, and two layers of leaf sheaths were gently collected at one time, upward, along the hypocotyl; if the cotyledons were unfolded, the most tender layer in the center of the cotyledons needed to be collected and immersed in sterile water, ensuring the uniformity and high quality of the materials used. The leaf sheaths were cut vertically with a razor blade and placed in mannitol. It was covered with tinfoil and left to stand in the dark at 26 °C for 10 min; the mannitol was then removed, the enzymatic solution was added, evacuated, and sealed with tinfoil, and the enzymatic digestion was carried out at low speed on a horizontal shaker for 4–5 h at 26 °C. At the end of enzymatic digestion, protoplasts were collected by a cell filtration sieve, washed once with W5 solution by a horizontal rotor for 3 min, and the final filtrate was resuspended to 5 mL. The supernatant was removed, leaving approximately 0.5 mL of precipitate, which was then resuspended in W5 solution. The composition of the W5 solution was as follows: weigh 9 g of NaCl, 17.25 g of CaCl₂, 0.37 g of KCl, 0.3 g of MES, and 0.9 g of glucose; add a part of ddH₂O, and then adjust the pH to 5.7 with 1 mol/L KOH solution; add ddH₂O to 1 L; sterilize at high temperature and autoclave; and store at 4 °C after 0.22 μm pore size suction filtration.

2.3. Plasmid Transfection of PEG-Mediated Protoplasts

Rice protoplast cells to be transformed were centrifuged and resuspended in MMg solution in 3 mL. Then, 10 μL of the OsRLCK118-1300GFP (concentration > 1000 ng/μL) plasmid and 100 μL of rice protoplasts were added to a sterile 2.0 mL centrifugal tube and gently mixed. After 10 min, the transformation was terminated by adding 440 μL of W5 solution, and the centrifuge tube was gently turned up and down until there were no obvious ripples. Centrifuge the tube in a horizontal rotor at room temperature, gently remove the supernatant, retain about 5 mL of liquid, add 1 mL of W5 solution to the wall, mix by turning the tube up and down, and repeat once. Aspirate the protoplasts into cell culture plates that were previously moistened and washed with 0.1% BSA, and incubate overnight (16 h) in low light, in a low-speed horizontal shaker at 26 °C and 50 rpm. After overnight cultivation, the fluorescence of the transformed body can be observed through fluorescence microscopy.

2.4. Immunoprecipitation and Mass Spectrometry Analysis (IP-MS)

The protoplasts transformed by centrifugation in a horizontal rotor at room temperature were gently removed from the supernatant, and 5 mL of buffer (50 μL PIC, 5 μL DTT, 15 μL Triton-100) was added for protein extraction from the protoplasts. After vortexing, the mixture was incubated on ice for 10 min and then centrifuged at 14,000 rpm for 10 min at 4 °C. Before use, anti-GFP affinity beads were washed twice with 1× PBS buffer. Equal amounts of total protein extract were added to two separate 1.5 mL centrifuge tubes: one for the experimental group (expressing the bait-GFP fusion protein) and the other for the negative control group (expressing the GFP empty vector). An equal amount of pre-washed anti-GFP beads was added to each tube. The mixtures were incubated on a rotator at 4 °C for 1–2 h to allow for sufficient protein–bead binding, followed by three to four stringent washes with lysis buffer at 4 °C (with low-speed centrifugation after each wash to collect the beads) to thoroughly remove non-specifically bound proteins. Finally, the supernatant was completely removed, and the bead pellet was retained. All the steps described above were performed in parallel under identical conditions. Eighty microliters of SDS loading buffer was added to the beads and heated at 95 °C for 8 min to elute the bound proteins. The eluate was used for subsequent Western blot validation and mass spectrometry analysis, which were also carried out under identical parallel conditions. We transferred SDS-PAGE gel to PVDF membrane, voltage 90 V, and ran it for 100 min. After transferring the membrane, we sealed it overnight at 4 °C in blocking solution (5% skim milk powder in 1× TBST). We added (1, 5000) 3 μL of primary antibody, 5% skim milk powder, and 1× TBST, and then we incubated overnight at 4 °C. After incubation, the membrane was washed three times with 1× TBST. Then, we added secondary antibody, 15 mL TBST + 0.45 g dry milk + (1, 10,000) 1.5 μL secondary antibody and washed three times with 1× TBST. Finally, we added HRP reaction substrate and incubated for 5 min. Then, we exposed and developed. The remaining supernatant was used directly for mass spectrometry sample preparation. To ensure data reliability, we applied stringent quality filters to the IP-MS data. Peptide-spectrum matches (PSMs) were accepted only if they met a Percolator q-value ≤ 0.01 (FDR < 1%) and XCorr thresholds. Proteins were considered identified if they contained at least one unique peptide and exhibited an experiment-wide q-value ≤ 0.01. For candidate selection, proteins were retained as high-confidence interactors only if they were present in the OsRLCK118-GFP pull-down samples and were detected in at least two out of three biological replicates. The IP-MS experiment was performed in three independent biological replicates. Mass spectrometry analysis was conducted by the Analytical and Testing Center of Fujian Agriculture and Forestry University.

2.5. PBT3-STE-OsRLCK118 Bait Gene Self-Activation and Toxicity Assay

The construction of decoy gene vectors for membrane systems was selected based on the fact that the decoy proteins were localized in different cellular compartments. To examine whether there is a signal sequence for such a localization, an online protocol at the website: https//services.healthtech.dtu.dk (accessed on 6 March 2025) and https://services.healthtech.dtu.dk/services/TMHMM-2.0/ (accessed on 6 March 2025) was used for prediction. The results showed that there was no discernible signal peptide at the N-terminus of OsRLCK118, and it is a β-barrel membrane protein anchored to the cell membrane via its N- or C-terminus. Thus, the bait gene, OsRLCK118-PBT3-STE, was constructed. Yeast two-hybrid screening was performed using the DUAL membrane activation kit, followed by subsequent testing according to the manufacturer’s instructions (Dualsystems Biotech, Zurich, Switzerland). As the DUAL membrane system can be used to screen full-length integral membrane proteins, cDNAs of the entire proteins were constructed and cloned by homologous recombination. The membrane protein system library in yeast strain NMY51 was purchased and activated on YPDA plates for 3 d. Using LiAc co-transformation, the following bait vector/control pairs were co-transfected into yeast competent cells, OsRLCK118-PBT3-STE and pPR3-N empty plasmid, OsRLCK118-PBT3-STE and pOst1-NubI plasmid, the positive bait plasmid pTSU2-APP and the positive prey plasmid pNubG-Fe65, and negative bait plasmid pTSU2-APP and negative prey plasmid pPR3-N. The transformants were added dropwise to the respective deficient plates SD-Trp-Leu, SD-Trp-Leu-His, and SD-Trp-Leu-His-Ade, and cultured in the incubator at 30 °C for about 4 d.

2.6. Quality Assessment of Yeast Libraries in Membrane Systems

The membrane library plasmid (1 μL) was electroporated into E. coli competent cells for amplification. Separately, 10 μL of the yeast library plasmid mixture was collected and serially diluted 10-fold, 100-fold, and 1000-fold with ddH₂O. A 50 μL aliquot from each dilution was plated on LB agar supplemented with ampicillin. After overnight incubation, monoclonal colonies were counted to determine the library transformation efficiency. Twenty random clones were then subjected to colony PCR, and the amplification products were analyzed by agarose gel electrophoresis to assess quality.

2.7. OsRLCK118-PBT3-STE Two-Hybrid Library Screening

The bait vector OsRLCK118-PBT3-STE was co-transfected into Saccharomyces cerevisiae yeast strain NMY51 and spread on SD-Leu plates for 2–3 d. Then, 2–3 full yeast monoclones were selected and transferred into 10 mL liquid medium (SD-Leu) at 30 °C, 220 rpm, 8 h shaking culture, and then transferred into 100 mL liquid medium (SD-Leu) at 30 °C, 220 rpm, 12–14 h shaking culture. Then, it was transferred to 100 mL liquid medium (SD-Leu), 30 °C, 220 rpm, 12–14 h shaking culture. After collecting the organisms with 50 mL, we resuspend them in 200 mL 2× YPDA liquid medium, 30 °C, 220 rpm, 4–5 h. After collecting the organisms with 700 g, 5 min, the yeast sensory state containing bait proteins was prepared by LiAc/TE, and the 7 μg library plasmid was transferred into the post-constructed reaction, and after expanding the culture, the organisms were resuspended with 0.9% NaCl, plated onto SD-Trp-Leu-His, and incubated in a 30 °C incubator for 4–5 d. Full yeast monoclonal clones were selected and streaked on an SD-Trp-Leu-His-Ade plate containing 20 mg/L X-α-Gal and incubated in a 30 °C incubator for 5 d. Positive colonies turned blue.

2.8. Extraction of Positive Yeast Plasmid and Plasmid Identification

We then picked the blue colony to be detected, in 800 μL YPDA liquid medium, 30 °C, 220 rpm, 8 h shaking culture. Then, we aspirated 300 μL of bacterial liquid into 5 mL YPDA liquid medium, 30 °C, 220 rpm, 16 h, and the remaining 500 μL of bacterial liquid was stored at −80 °C. Yeast plasmid extraction method was referred to the yeast plasmid extraction kit of Zhongke Ruitai Biotechnology company (Beijing, China). The plasmid was amplified using the identification primers (Table S1). The recoveries were identified and sequenced by agarose electrophoresis, and the sequencing results were compared on the RIGW website. The candidate genes encoding known proteins were compiled and statistically analyzed, with duplicates removed to obtain the final results.

2.9. Yeast Plasmid Amplification

The yeast plasmid was transferred to the E. coli DH5α on an LB plate (containing ampicillin) and incubated at 37 °C overnight. Each single clone was picked to a 5 mL liquid LB (containing ampicillin) and incubated at 37 °C, 220 rpm shaking for 16 h. Escherichia coli (E. coli) plasmid extraction method refers to Zhongke Ruitai Biotechnology company common plasmid mini-extraction kit SOP; the plasmid obtained was used for one-to-one MYTH interactions validation.

2.10. Interaction Verification Between Prey Plasmid and Bait Plasmid

The positive control is pTSU2-APP + pNubG-Fe65. The negative controls are PBT3-STE + SAMS1-pPR3-N and PBT3-STE-OsRLCK118 + pPR3-N. The experimental group, PBT3-STE-OsRLCK118 + SAMS1-pPR3-N, co-transfected the above plasmids into NMY51, incubated in a water bath at 42 °C for 45 min, centrifuged at 700 rpm for 5 min, and then resuspended the bacterial cells in 0.9% NaCl. Plated on SD-Trp-Leu medium plates and incubate at 30 °C for 3 d. After growth, we took a complete single clone and cultured it in 100 μL of 0.9% NaCl solution at 30 °C for 1 h. After that, we took 15 μL of SD-Trp-Leu and SD-Leu-Trp-His-Ade culture medium plates, incubated at 30 °C for 3–5 d, and then observed the results.

2.11. Data Set Analysis

In order to effectively analyze and compare the IP-MS identification results with the membrane system yeast two-hybrid (MYTH) screening results and to derive the various classification statistics, we used the following steps for dataset analysis. First, we collated a list of all proteins identified by the IP-MS, including information such as protein names, identification scores, coding proteins, etc. The data were obtained from the National Rice Database Center (NRDC). For the membrane system yeast library dataset, we similarly collated a list of all the proteins screened by this method, including protein name, coding protein, and other information, with data from the NRDC: https//www.ricedata.cn/gene/ (accessed on 25 August 2024). The data were further categorized and summarized in terms of biological processes, molecular functions, and cellular composition through the website http//www.geneontology.org/conducted (accessed on 10 December 2024). GO analysis data were organized and analyzed for graphing using the Venny tool (https//bioinfogp.cnb.csic.es/tools/venny/) (accessed on 10 December 2024).

2.12. Split-Luciferase Complementation Assay

In order to verify the complementation of the two genes to be detected, we first cloned their CDSs (coding sequences) into the pCAMBIA1300 vector using homologous recombination to construct the fusion expression plasmids of NLuc and CLuc, respectively. Subsequently, these two plasmids were introduced into Agrobacterium GV3101, respectively, and screened on LB plates containing kanamycin and rifampicin, which were placed in a 28 °C incubator for 2 to 3 days. The monoclonal colonies were picked from the plates and first subjected to small-scale liquid culture (4–6 h) and initially identified by colony PCR. After confirmation, the positive clones were expanded and cultured in 5 mL of LB liquid medium containing kanamycin and rifampicin, and then they were incubated for 12 to 16 h at 28 °C and 220 rpm with shaking. At the end of the incubation, the organisms were collected by centrifugation (4000 rpm, 10 min), followed by resuspension of the organisms with freshly prepared Nicotiana benthamiana injection and adjustment of the optical density (OD) of the organisms to a range of 0.8 to 1.2. After the resuspended bacterial solution was allowed to stand for 4 h, the bacterial solution was injected into the leaves of Nicotiana benthamiana using a 1 mL syringe. The injected tobacco plants were incubated in a greenhouse for 48 to 72 h, after which the leaves at the injection site were removed. Fluorescein (Beetle Luciferin, Promega product) at a concentration of 100 mmol/mL was evenly applied to the back of the leaf and darkened for 10 min. Finally, the leaves were imaged using an in vivo imager to visualize the fluorescence signals in order to assess the interactions between the genes.

2.13. Co-IP Assay

We selected the binding beads according to the label of the target protein and added 25 μL of beads for every 1 mL of protein sample. The beads were then suspended in 1 mL of pre-cooled PBS and centrifuged at 2000× g for 2 min; this washing procedure was repeated three times. Subsequently, the protein sample was introduced to the beads and incubated at 4 °C for 3–4 h under gentle agitation. Following incubation, the mixture was centrifuged at 4000× g for 2 min, and the supernatant was carefully removed. The beads were then resuspended in 1 mL of pre-cooled PBS supplemented with 0.1% Triton X-100, centrifuged again at 2000× g for 2 min, and subjected to four additional washing cycles. The beads were resuspended in 80 μL of PBS. Then, 20 μL of 5× SDS loading buffer was added, and the mixture was heated at 95 °C for 10 min in a heat block, followed by brief cooling on ice for 5 min.

2.14. In Vitro Kinase Assay

A 30 μL in vitro phosphorylation reaction mixture was prepared on ice, containing 3 μL 10× kinase buffer (prepared with 20 μL 1 M Tris-HCl (pH 7.5), 10 μL 1 M MgCl₂, 10 μL 1 M CaCl₂, 1 μL 1 M DTT, and ddH₂O to a final volume of 100 μL), 3 μL 1 mM ATP, 8 μL kinase protein, and 16 μL substrate protein, with ddH₂O added to adjust the total volume to 30 μL. After gentle mixing, the reaction mixture was incubated in a 30 °C metal bath for 40 min to allow for phosphorylation. Subsequently, 7.5 μL of 5× SDS loading buffer was added, followed by denaturation at 70 °C in a metal bath for 8 min. The sample was centrifuged at 12,000 rpm for 1 min to collect the supernatant. Finally, total protein was visualized by Coomassie Brilliant Blue (CBB) staining, and phosphorylated substrate signals were analyzed via Western blot (WB).

2.15. Western Blot (WB) Experiment

Proteins were separated on a 10% SDS-PAGE, first concentrated at 90 V for 30 min and then separated at 120 V for 90 min. After electrophoresis, the gel was transferred onto a PVDF membrane (pre-activated in methanol) using a wet transfer system (100 V, 90 min) in transfer buffer, with the assembly arranged in the order of anode–sponge–filter paper–gel–PVDF membrane–filter paper–sponge–cathode. The membrane was then blocked with 5% skim milk in 1× TBST for 1–2 h at room temperature, followed by incubation with the corresponding primary antibody diluted in 5% skim milk/TBST overnight at 4 °C. After three washes with TBST (5 min each), the membrane was incubated with the appropriate HRP-conjugated secondary antibody in 3% skim milk/TBST for 1 h at room temperature with gentle shaking. The primary antibodies used in this study were obtained from Abmart and related companies, all applied at a dilution of 1:5000 in 5% skim milk/TBST. The α-pSpT primary antibody was detected using a horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibody, whereas the α-GFP, α-HA, and α-Cluc primary antibodies were detected using an HRP-conjugated goat anti-mouse secondary antibody. All secondary antibodies were diluted at 1:10,000 in 3% skim milk/TBST.

2.16. Spray Inoculation with M. oryzae (Guy11)

In the inoculation experiment with M. oryzae, spores were scraped from the surface of a straw rice bran medium using sterile slides (composition of rice bran medium: 40 g of rice bran, 20 g of agar powder, and 300 μL of 1 mol/L potassium hydroxide). After adding ultrapure water to bring the volume to 1 L, we then washed and filtered with a solution containing 0.02–0.15% Tween-20, and the spore concentration was adjusted to 1 × 10⁵ per/mL. The Guy11 fungus was provided by the laboratory of Prof. Dingzhong Tang, Plant Immunity Center, Fujian Agriculture and Forestry University. Healthy rice seedlings that were 15–21 days old were selected and immediately incubated in darkness at 25–26 °C and 80% humidity for 24 h after uniform spray inoculation, and then they were transferred to an artificial climate room with photoperiod control, temperature 26 ± 1 °C, and humidity ≥ 80% to continue cultivation. The leaves were atomized and kept wet every day. Systematic disease observation and phenotype recording were conducted within 3–7 days after inoculation. All disease-resistance phenotype analyses (such as lesion counts and disease index) used individual rice plants as independent biological replicates. For each genotype, at least n = 12–15 independently grown plants were included. All experiments were performed in ≥3 independent biological replicate trials, with consistent trends observed across replicates. For count data such as lesion numbers, appropriate non-parametric statistical tests were applied for analysis, while data conforming to a normal distribution were analyzed using analysis of variance (ANOVA).

3. Results

3.1. IP-MS Screening of OsRLCK118-Interacting Proteins

To identify proteins that interact with OsRLCK118 in vivo, the OsRLCK118-GFP fusion construct was transformed into rice protoplasts. The expression of the fusion protein was confirmed by SDS-PAGE, showing a band at the expected molecular weight (Figure S1A). The interacting target proteins were enriched through immunoprecipitation assays with an anti-GFP antibody and subsequently identified using IP-MS.

A total of 881 proteins were identified by the OsRLCK118-GFP fusion protein pull-down, and 134 proteins were identified in the GFP-only control. After removing the duplicated negative control, the OsRLCK118-GFP pull-down specifically enriched a total of 781 proteins (Figure 1A,B; Tables S2–S5). A complete list of all identified proteins, along with detailed quantitative metrics, including enrichment ratios, replicate coverage, unique peptide counts, spectral counts, and q-values, is available in Tables S2–S5. These proteins were categorized according to the gene description provided by the National Rice Data https://www.ricedata.cn (accessed on 25 August 2024). Among them, ribosome-associated proteins were the most numerous, accounting for 19.5% (152) of the total proteins. This is likely due to the fact that in GFP-tagged IP-MS analysis, highly expressed housekeeping proteins are prone to nonspecific adsorption, thereby constituting the primary background detected with significant enrichment. Transporter proteins and membrane proteins accounted for 13.7% (107) of the total, including carrier protein and aquaporin, are responsible for the intra- and extracellular or organelle-to-organelle transport. From a biological and functional perspective, this enrichment holds potential rationale and biological significance. As a cytoplasmic kinase, OsRLCK118-mediated immune signal transduction likely relies on precise membrane localization and transmembrane transport of substances.

The third category was metabolism and enzyme activity, with a total of 102 (13%), of which oxidoreductases (LOC_Os08g33720, etc.) and metabolism-related proteins (LOC_Os04g58110, LOC_Os02g07260, etc.) may be associated with plant disease resistance to some extent. For example, OsAPX2 (LOC_Os07g49400) is ascorbate peroxidase that was shown to positively regulate rice blast resistance [23]. OsPT8 (LOC_Os10g30790), a phosphate transporter, regulates phosphate (Pi) signaling for development and negatively regulates rice immunity [24].

Additionally, 60 proteins involved in protein synthesis, degradation, and modification were identified. It is noteworthy that several proteins related to ubiquitination, such as OsGF14e (LOC_Os02g36974), which plays an important role in rice defense response and tolerance to abiotic stresses, negatively affecting the induced expression of rice defense response genes, cell death, and resistance to leaf blight and stripe blight [25,26]. It may act downstream of OsRLCK118 and play a role in disease resistance through ubiquitination.

There are 48 proteins in the signaling class, including ethylene-insensitive OsEIL1 (LOC_Os03g20790), which acts downstream of OsEIN2 and regulates ethylene responses in rice roots [27]. The ethylene signaling pathway may be involved in environmental humidity regulation of rice blast development, and rice blast fungus infestation induces ethylene synthesis of OsEIL1 [28]. OsRac5 (LOC_Os01g12730), small GTPase; small GTP-binding protein, which interacts with OsRbohB; and the OsRac5-OsRbohB module generate ROS, modulating the immune response of rice [29].

There were 44 each of transcriptional and translational regulatory class proteins and stress response and defense proteins, where stress response and defense included biotic/abiotic stress (LOC_Os04g58090, LOC_Os11g06720, etc.), oxidative stress (LOC_Os07g08840, LOC_Os04g33970, etc.), and pathogen defense (LOC_Os09g27260, etc.). In plant disease resistance, OsHin1 (LOC_Os04g58850) is a harpin-induced gene, and harpin is an exciton produced by plant pathogens that induces a disease-resistance response in plants [30]. There are 36 proteins in the kinase class. The main function of kinases is to catalyze the transfer of phosphate groups away from ATP to specific substrates, thereby regulating intracellular signaling, metabolic pathways, and so on. In rice disease resistance, kinases may be involved in processes such as recognition of pathogens and activation of defense responses. For example, OsRLCK176 (LOC_Os05g02020), a receptor-like cytoplasmic kinase, acts downstream of OsCERK1 to regulate chitin- and peptidoglycan (PGN)-induced immune responses, and it is essential for intracellular PGN and chitin signaling in rice [31]. Finally, there are 18 proteins related to photosynthesis and plant development, and finally the remaining proteases/peptidases (21), other functionally defined proteins (44), and unknown proteins (65).

The results of the GO functional annotation showed that the entries significantly enriched in differentially expressed proteins were classified into two main categories, biological process (BP), and molecular function (MF) (Figure 1C,D). Biological processes mainly include metabolic process, cellular process, and localization. Molecular function mainly includes binding, catalytic activity, transporter activity, and structural molecule activity.

3.2. Screening of OsRLCK118-PBT3-STE Interaction Proteins in Yeast

The enriched proteins obtained from IP-MS screening constitute a mixed pool comprising direct interaction targets, indirectly associated proteins, and non-specific background components. To identify the genuine targets that directly and specifically interact with OsRLCK118, we performed membrane yeast two-hybrid (MYTH) screening assays. Firstly, the positive controls (pTSU2-APP + pNubG-Fe65) exhibited normal growth on both SD-Trp-Leu and SD-Leu-Trp-His-Ade selective media, but the negative controls (bait vector OsRLCK118-PBT3-STE + pPR3-N) grew normally on SD-Trp-Leu medium and showed no growth on SD Leu-Trp-His-Ade medium (Figure 2A). This confirms the absence of self-activation activity in the bait protein OsRLCK118. Subsequently, the constructed cDNA library was transformed into E. coli, and 10-fold, 100-fold, and 1000-fold gradient dilutions were plated on LB medium containing ampicillin to assess the library titer and transformation efficiency. Validation tests confirmed that the library exhibited a recombination rate of 100%, with an average insertion fragment greater than 1000 bp (Figure 2B,C), meeting the screening criteria.

Then, we used this bait carrier to screen the prey protein in the yeast membrane system and obtained 708 positive colonies from the membrane cDNA system (Figure 2D,E). After confirmation of a small number of the yeast colonies with plasmid extraction, PCR detection was performed with the pPR3-N vector primer for all colonies, and the collectively amplified DNA was sent for DNA sequencing after agarose gel separation. The sequencing results were aligned using the Rice Information Gateway and analyzed by the China Rice Data Center (Table S6). From this screen, 402 genes encoding known proteins were initially obtained. After removing duplicates, 287 non-redundant genes encoding known proteins were ultimately identified, with a false-positive rate of approximately 56.7%.

GO annotation analysis showed that the proteins encoded by these 287 genes are involved in biological processes such as cellular metabolic processes, reproductive processes, cellular developmental processes, localization of cellular components, activation and qualification of self-stabilizing processes, cellular growth and physiological processes, and interactions (Figure 2F). Among them, OsMNAC3 (LOC_Os07g48450), the NAC transcription factor, negatively regulates rice blast and leaf blight resistance in rice, as well as PTIs by PAMP [32]. The 14-3-3 protein, GF14c (LOC_Os08g33370), directly interacts with and regulates the stability of OsSCL7, thereby positively regulating the rice immunity, and knockdown of GF14c increased the susceptibility of rice to M. oryzae [33]. OsGns6/PR2 (LOC_Os01g71350) and PR3 (LOC_Os06g51050) are pathogenesis-related genes, which are catalytically active, and rice blast fungus infestation induces Gns6/PR2 expression [34] (Wang et al., 2021). Moreover, OsPR3 modulates immune responses in rice [35].

Some of the proteins related to binding could also be involved in rice disease resistance. For example, OsGRF8 is a target gene of OsmiR396 that directly regulates the flavanone 3-hydroxylase gene, OsF3H (LOC_Os03g03034). OsSBP (LOC_Os01g68770) is a selenium-binding protein homologue gene, and it has been found to regulate Botrytis cinerea infestation [36] (Figure 2G).

Cellular component analysis indicated that the proteins were localized to membranes, the cytoplasm, and the nucleus. These components play crucial roles in rice disease resistance. They not only provide structural and functional support to rice cells but also potentially participate in a variety of biological processes related to disease resistance (Figure 2H).

3.3. Common Proteins Found in Both IP-MS Identification and Yeast Library Screening

IP-MS results reflect the protein interaction network potentially associated with OsRLCK118 under near-physiological conditions (including both direct and indirect interactions), whereas membrane yeast two-hybrid assays are more adept at detecting direct binary interactions [37]. Taking the intersection of these two datasets indicates that these proteins not only specifically associate with OsRLCK118 in a realistic cellular environment but also directly bind to it, thereby reducing the risk of false positives inherent to any single technique. By integrating the results from IP-MS and MYTH screening, we identified 35 overlapping high-confidence candidate interactors of OsRLCK118 (Figure 3A; Table S7), 10 of which are known immune-related proteins (Table S8). GO classification revealed distinct functional enrichment; at the cellular component level, proteins were predominantly localized to membrane structures. Among biological processes, metabolic processes were the most represented, with 10 proteins assigned to this category, and in molecular functions, the “catalytic” and “binding” activity functions were more frequent, encompassing a total of 19 proteins (Figure 3B).

3.4. OsRLCK118 Interacts with and Phosphorylates OsSAMS1

Among these 35 candidate proteins, we selected one protein for interaction based on our literature research, functional prediction, and experimental feasibility. OsSAMS1 is an S-adenosyl-L-methionine synthase that is positively correlated with plant ethylene synthesis and plays an important role in abiotic stress. Transcriptome analysis in our laboratory showed that the expression level of OsSAMS1 gene significantly increased after induction by M. oryzae, indicating that OsSAMS1 is involved in the immune response of rice and may regulate resistance to M. oryzae [38]. Similarly, OsRLCK118 is also a positive regulator of rice resistance [19]. In terms of mechanistic novelty, established precedents in Arabidopsis demonstrate that RLCK family members can regulate ethylene signaling pathways through phosphorylation. This provides a direct and robust molecular basis for exploring a novel “kinase-primary metabolic enzyme” immune regulatory model in rice through the interaction between OsRLCK118 and OsSAMS1 [39,40,41]. Furthermore, OsSAMS1 itself lacks kinase activity, which facilitates subsequent validation of whether OsRLCK118 can phosphorylate and modify OsSAMS1 to regulate its activity. Therefore, we designed a luciferase complementation assay (split-LUC), a membrane system yeast two-hybrid assay, and Co-IP to further validate the interaction between OsSAMS1 and OsRLCK118.

A split-LUC in tobacco leaves showed strong luminescent signals when OsRLCK118-NLuc and OsSAMS1-CLuc were co-expressed, while control combinations yielded no fluorescence signal, indicating a specific interaction in planta (Figure 4A). The expression of all fusion proteins was verified by Western blot (Figure S1B), ensuring that the observed luminescence specifically reflected protein–protein interaction. Further validation using MYTH assay demonstrated that co-expression of OsRLCK118-PBT3-STE and OsSAMS1-pPR3-N enabled yeast growth on selective media, similar to the positive control, whereas negative controls failed to grow, supporting their interaction in yeast cells (Figure 4B). Finally, Co-IP assays in tobacco cells revealed the presence of both OsRLCK118 and OsSAMS1 in immunoprecipitated complexes, confirming their interaction in a physiological context (Figure 4C). These results demonstrate a specific and functional interaction between OsRLCK118 and OsSAMS1 across both heterologous and native experimental systems.

In plants, phosphorylation is an important way of protein post-translational modification. Phosphorylation/dephosphorylation can regulate protein activity, stability, cell localization, etc., participating in the regulation of various biological processes [42]. In rice, OsRLCK118 has been confirmed to positively regulate rice blast resistance, and OsRLCK118 may trigger a series of immune responses by sensing external pathogen signals to enhance rice blast resistance [18]. RLCKs function via phosphorylation of downstream target proteins and are mainly involved in physiological processes such as plant growth, signal transduction, abiotic stress, and biological stress response [19].

Since OsRLCK118 is a kinase, we investigated whether OsSAMS1 might serves as its substrate. To test this, we designed an in vitro phosphorylation assay by co-incubating OsRLCK118 and OsSAMS1 in an ATP-containing buffer, with control groups containing only OsRLCK118 or OsSAMS1. The results showed that a phosphorylation signal was detected in the control containing OsRLCK118 (indicating its autophosphorylation activity), but no signal was observed in the control containing only the substrate OsSAMS1. However, upon the simultaneous addition of both the kinase and the substrate, a clear phosphorylation signal was detected for the substrate, indicating that OsSAMS1 can be phosphorylated by OsRLCK118 in vitro conditions. (Figure 4D).

In summary, the interaction between OsRLCK118 and OsSAMS1 was consistently demonstrated by split-LUC assay, MYTH assay, and Co-IP validations, and such an interaction is the basis for OsRLCK118 to exert its kinase activity on the substrate OsSAMS1. Meanwhile, OsRLCK118-mediated phosphorylation of OsSAMS1 may be a component of the immune signaling network.

3.5. OsSAMS1 Acts Downstream of OsRLCK118 in Rice Blast Resistance

To further dissect the immune regulatory mechanisms in rice and provide genetic evidence, we used CRISPR/Cas9 technology to knock out the OsRLCK118 gene in the rice variety ZH11. Two 20 bp sgRNA target sites were designed within its coding sequence (CDS). Sequencing confirmation led to the establishment of two independent knockout lines, designated osrlck118 (Figure 5A,B). The ossams1 mutant was previously generated and provided by our laboratory [38]. Subsequently, the double mutant osrlck118-ossams1 was successfully constructed through genetic hybridization, and then it was inoculated with M. oryzae Guy11 fungus to analyze its genetic relationship. The results showed that compared with the wild-type ZH11, both osrlck118 and ossams1 single mutants exhibited similar increased susceptibility (Figure S2). More importantly, the double mutant osrlck118-ossams1 showed no significant difference in lesion number compared to the single mutant ossams1, suggesting that ossams1 and osrlck118 are in the same linear pathway, and osrlck118 may be upstream of ossams1 (Figure 5C,D).

4. Discussion

4.1. Identification of Candidate Proteins Associated with OsRLCK118 Interactions

Through IP-MS and yeast membrane system screening, we identified 35 proteins with high interaction potential with OsRLCK118. Based on their functional characteristics, we categorized them into the following three classes:

Signal transduction-related proteins: The 14-3-3 protein GF14f may function by binding to the phosphorylation site of OsRLCK118 to regulate its activity and participate in disease-resistance responses [43], the small G-protein OsRab7B3 may cooperatively regulate vesicle transport and signal transduction [44], and the transcriptional cofactor OsHMGB1 may be involved in the regulation of gene expression related to phosphate signaling [45].

Stress response-related proteins: Ascorbate peroxidase OsAPX2, as a key regulator of ROS homeostasis, may enhance the plant’s antioxidant capacity through its interaction [46], and the xyloglucanase inhibitory protein HI-XIP may jointly participate in cell wall-related stress responses [47].

Substance transport and metabolism-related proteins: The acyl-CoA-binding protein OsACBP4 may reveal a novel function of OsRLCK118 in fatty acid metabolism regulation [48], the phosphate transporter accessory protein OsPHF1 may cooperatively regulate phosphorus balance [49], and the tonoplast intrinsic protein OsTIP1 may be involved in water balance regulation [50].

The discovery of these interacting proteins provides important clues for elucidating the molecular mechanisms of OsRLCK118 in signal transduction, metabolic regulation, and stress adaptation.

4.2. Biological Significance of OsRLCK118 and SAMS1 Interactions

The OsRLCK118–OsSAMS1 interaction was established through a progressive evidence chain: initial discovery from IP-MS and MYTH screens; orthogonal validation by split-LUC and co-IP assays; and functional correlation via phosphorylation, ethylene measurement, and genetic analyses. This cumulative approach supports OsSAMS1 as a bona fide interactor and potential effector of OsRLCK118 in rice immunity.

Furthermore, our initial in vitro phosphorylation assay suggests that OsSAMS1 may serve as a putative substrate of the OsRLCK118 kinase. This discovery provides key molecular clues for understanding how OsRLCK118 enhances rice disease resistance, potentially by linking kinase signaling to the regulation of ethylene synthesis. It also suggests a novel pattern of synergy between immune kinases and metabolic enzymes. The current research, however, remains at a preliminary stage. To definitively confirm the directness and specificity of this phosphorylation event, future studies are required to incorporate key controls in the in vitro assays, such as kinase-dead mutants and ATP-minus reaction systems. Therefore, while our results indicate a biochemical interaction between these two proteins, they do not conclusively establish direct and specific phosphorylation causality. The interpretation of OsSAMS1 as a direct substrate of OsRLCK118 should thus be considered preliminary. Furthermore, it is necessary to validate whether this phosphorylation modification genuinely occurs under plant physiological conditions through in vivo experiments, for instance, by immunoblot analysis using phosphorylation-specific antibodies.

4.3. The Functional Significance of OsRLCK118 Phosphorylating OsSAMS1

SAMS1 is a key enzyme that catalyzes the synthesis of S-adenosyl-L-methionine (SAM). As a methyl donor in organisms, SAM plays an important role in multiple biological processes, such as DNA methylation, histone methylation, and biosynthesis [51,52]. In rice, OsSAMS1 has been confirmed to be involved in regulating leaf senescence, seed germination, and other processes, and positively regulating blast resistance in rice [53]. OsSAMS1 may regulate these biological processes by influencing ethylene synthesis [54,55]. Ethylene, as an important plant hormone, as it plays a wide role in plant growth, development, and stress response, and SAMS1 is involved in ethylene synthesis. Ethylene levels are elevated in plants overexpressing SAMS1 [53,56].

Phosphorylation of OsSAMS1 by OsRLCK118 may enhance its enzyme activity and further promote the accumulation of SAM, providing an adequate supply for ethylene synthesis. This mechanism is consistent with findings in previous studies: the loss of OsSAMS1 function leads to the downregulation of ethylene synthesis genes (such as OsACS2) and a significant reduction in blast resistance [38]. To directly test this regulatory hypothesis, we will integrate the proposed material construction and phenotypic analysis strategies. Specifically, upon generating the stable phosphorylation-deficient (S/T → A) and phosphorylation-mimetic (S/T → D/E) OsSAMS1 rice lines, we will subject these materials—along with wild-type and relevant mutant controls—to M. oryzae infection and subsequent molecular phenotyping. In addition to assessing the expression of ethylene synthesis genes via qPCR, we will employ gas chromatography–mass spectrometry (GC–MS) to quantify SAM and ethylene levels in these genetic backgrounds. This combined approach will enable us to evaluate whether phosphorylation of OsSAMS1 indeed enhances its enzymatic output in vivo, elevates SAM and ethylene accumulation, and consequently strengthens blast resistance. In addition to serving as the source of SAM, the precursor for ethylene synthesis, SAM also functions as a methyl donor for methyltransferase reactions. Therefore, the phosphorylation of OsSAMS1 may also regulate immune responses by influencing the methyl cycle and downstream methylation modifications. This mechanism is not mutually exclusive with the ethylene pathway; instead, they may together constitute a complex regulatory network.

Secondly, phosphorylated OsSAMS1 may affect protein stability. Phosphorylation may extend the half-life of OsSAMS1 by inhibiting the ubiquitination-mediated degradation pathway, maintaining its continuous function. Meanwhile, it can also be verified by combining subcellular localization in cells. To directly test this hypothesis, we plan to conduct systematic experiments to investigate the impact of phosphorylation on OsSAMS1 stability and subcellular localization. Specifically, we will employ site-directed mutagenesis to generate phosphorylation-inactive (S/A) and phosphorylation-mimetic (S/D) mutants of OsSAMS1. The protein expression levels of OsSAMS1 in plants will be detected using WB to validate the effect of phosphorylation on its protein stability. Meanwhile, regarding the potential effect on localization, we plan to construct fluorescent protein fusion vectors of these mutants to observe their subcellular localization via confocal microscopy in rice protoplasts or transgenic plants. This will allow us to verify whether the subcellular localization of OsSAMS1 changes due to phosphorylation, potentially enabling it to function more efficiently in compartments relevant to ethylene synthesis, such as the cytoplasm or endoplasmic reticulum. This potential change in localization and its functional implications remain to be experimentally tested through the proposed strategies.

4.4. The Synergistic Mechanism Between Ethylene Signaling Mediated by Phosphorylation and Disease Resistance

In plants, it has been found that ethylene is involved in plant disease resistance. After sensing the pathogen PAMP factor, plants can rapidly synthesize ethylene within the plant to resist the invasion of pathogens [57]. At the same time, ethylene can combine with endogenous peptides to amplify the PTI reaction signal of plants, leading to persistent resistance in plants [58]. In rice, ethylene has also been shown to play a very important role in disease resistance. For example, rice has evolved broad-spectrum disease-resistance NLR receptors that can inhibit the interaction between pathogenic virulence proteins and PICI1, thereby protecting and enhancing the function of PICI1. This, in turn, activates the synthesis of more defense-related chemicals (methionine–ethylene), leading to broad-spectrum disease resistance [1]. Overexpression of OsACS2, a key enzyme gene encoding ethylene synthesis, significantly enhanced rice blast resistance [59]. Increasing ethylene content in rice can improve blast resistance [60], and silencing OsEIN2b, the central transmitter of ethylene synthesis gene, significantly reduces blast resistance [61]. The expression of genes related to ethylene synthesis was significantly inhibited in OsSAMS1 knockout line, and the resistance to rice blast was significantly decreased in the knockout line.

Although we initially validated the interaction and phosphorylation relationship between OsRLCK118 and OsSAMS1, in order to further investigate their functional dependence, we created the double mutant osrlck118-ossams1. The identification of resistance to rice blast disease showed that compared with the wild type, the resistance of osrlck118 and ossams1 mutants was significantly reduced, and the lesion area was significantly increased. Importantly, genetic analysis revealed that while the ossams1 single mutant was highly susceptible, the ossams1-osrck118 double mutant did not show a significant increase in lesion number. This lack of additive effect suggests that OsSAMS1 and OsRLCK118 function in the same genetic pathway. Therefore, OsSAMS1 is likely a key downstream component required for OsRLCK118-mediated disease resistance. In theory, if the contribution of OsSAMS1 to rice blast resistance is completely independent of OsRLCK118, then the dual mutant phenotype should represent a combination of two single mutant phenotypes. Furthermore, based on the observed epistatic relationship—where OsSAMS1 functions downstream of OsRLCK118—our next step is to complement the osrlck118 mutant with wild-type OsSAMS1. This approach will provide direct genetic evidence to confirm this directional regulation. If OsSAMS1 acts strictly downstream in this pathway, such complementation is expected not to restore the disease-resistant phenotype in the osrlck118 background.

Next, focusing on the verification of phosphorylation sites and functions, we will use mass spectrometry and site-directed mutagenesis to verify the specific phosphorylation sites of OsSAMS1, use CRISPR-Cas9 technology to accurately knock out OsSAMS1 in rice, and based on this, upregulate the expression of wild-type OsSAMS1, phosphorylation-inactivated (S/A), and phosphorylation-mimicked (S/D) mutants. In addition to analyzing their effects on enzyme activity and protein stability, it is also necessary to clarify whether OsRLCK118 can directly regulate PR gene expression through the ethylene synthesis pathway. Future research should employ transcriptomic analysis (e.g., RNA-seq) or qPCR to directly compare the expression patterns of classic PR genes (such as WRKY45 and PAL) in wild-type plants, mutants, and complemented lines with different phosphorylation forms, thereby establishing a causal relationship between ethylene signaling burst and the activation of defense genes.

In conclusion, this study identifies OsSAMS1 as a candidate downstream effector of OsRLCK118 and proposes a hypothetical working model in which OsRLCK118 phosphorylates OsSAMS1 to enhance ethylene biosynthesis and disease resistance. This model is derived from our current dataset, including biochemical interaction, in vitro phosphorylation, and genetic epistasis analyses. But it should be interpreted as a testable framework rather than a definitive mechanism. The discovery of the OsRLCK118-OsSAMS1 regulatory module not only deepens our understanding of the functional diversity of the RLCK family but also provides new clues for designing novel disease-resistance strategies based on kinase–metabolic enzyme interactions. Future research needs to combine multi-omics and molecular design breeding to optimize the expression patterns of sRLCK118/OsSAMS1 and cultivate rice varieties with synergistic improvement of disease resistance and agronomic traits (Figure 6).

5. Conclusions

In summary, through integrated IP-MS and MYTH screening, we identified 35 high-confidence candidate interacting proteins of the rice immune kinase OsRLCK118. Among these, the ethylene biosynthesis enzyme OsSAMS1 was validated as a key interaction protein through multiple experimental approaches. Our data propose a hypothetical working model in which OsRLCK118 may enhance OsSAMS1 activity via phosphorylation to promote ethylene biosynthesis and thereby strengthen rice blast resistance against M. oryzae. This model is strictly derived from our current dataset, including biochemical interaction, in vitro phosphorylation, and genetic epistasis analyses.

The findings reveal a preliminary clue that RLCKs amplify immune signaling through metabolic reprogramming. Subsequent studies will further validate this model through experiments such as mapping the specific phosphorylation site(s) on OsSAMS1 targeted by OsRLCK118; generating and phenotyping phospho-dead (S/A) and phospho-mimetic (S/D) transgenic lines; and developing phosphorylation-specific antibodies to monitor OsSAMS1 phosphorylation in vivo under pathogen challenge.