Seeds of wild type (WT) rice Oryza sativa cv. Nipponbare and OsRIP1-OE plants (T4 generation) from line J and line H (Wytynck et al., 2021) were dehusked, and soaked in 70% ethanol on a shaker (130 rpm) for 5 minutes, followed by 45 minutes washing in 5% NaOCl solution containing 0.01% Tween-20. After extensive washing with sterilized H₂O seeds were incubated overnight in H₂O on a shaker (130 rpm) at 28°C.

Sterilized seeds were germinated in ½ solid MS medium (pH 5.8) supplemented with 30 g/l sucrose, 8 g/l Agarose SPI (Duchefa Biochemie, Netherlands) and 1.12 mg/l Gamborg B5 vitamins (Duchefa) in square Petri dishes, sealed with micropore tape. Seeds of transgenic plants from line J and line H were germinated on selective medium containing 4 mg/l phosphinothricin (Duchefa). Petri dishes were wrapped with aluminum foil and incubated in the dark in a plant chamber at 28°C. After 4 days, the aluminum foil was removed, and germinated seeds were grown at 28°C with a 16-h light/8-h dark cycle for an additional 3 days. One-week-old rice seedlings were grown hydroponically in the ½ Hoagland solution under the same conditions in a plant cabinet. The ½ Hoagland solution was refreshed daily. 14-day-old plants were photographed and the phenotypic differences between WT and OsRIP1-OE transgenic plants were analyzed for biomass, shoot length and root length.

14-day-old plants were subjected to MeJA treatment. MeJA (Sigma-Aldrich, Darmstadt, Germany) was dissolved in absolute ethanol (Sigma-Aldrich) to obtain a 100 mM stock solution, and then added to the ½ Hoagland solution to reach the working concentration of 100 μM. Control seedlings were kept in the ½ Hoagland solution with 0.1% (v/v) ethanol. Shoots and roots were sampled at 3 h and 24 h, and stored at -80°C. All treatments were set up for biological triplicates.

Four treatment groups were set up for each indicated timepoint (3 h and 48 h), namely mock-treated WT plants, MeJA-treated WT plants, mock-treated T4 OsRIP1-OE transgenic rice plants line J, and MeJA-treated T4 OsRIP1-OE transgenic rice plants line J. Three independent biological replicates were performed for each treatment, containing 10-12 individual plants per replicate. Total RNA was extracted from freshly ground material using the Spectrum Plant Total RNA kit (Sigma-Aldrich). Library preparation, sequencing and data analysis were performed as previously described (Ghaemi et al., 2020). For each time point, Gene Ontology (GO) enrichment analyses were performed for significantly differentially expressed genes (Adjusted P-values, FDR< 0.05, and |log₂-FC| > 1) using PLAZA 4.5 (https://bioinformatics.psb.ugent.be/plaza/versions/plaza_v4_5_monocots/).

The reverse-transcribed cDNA was synthesized using Maxima First-Strand Synthesis kit (Thermo Fisher Scientific, Waltham, Massachusetts, USA) after RNA extraction and DNAse treatment. Transcript levels for genes of interest were analyzed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) using gene specific primers ( Supplementary materials 1 , Supplementary Table S1 ). EXP, EXPNar and EIF5C were selected as the reference genes. RT-qPCR was performed using the 96-well CFX Connect™ Real-Time PCR Detection System (BioRad, Hercules, California, U.S.) with iQ™ SYBR^® Green Supermix (BioRad). The PCR amplification steps were 95°C for 10 min, followed by 41 cycles of 95°C for 15 sec, 60°C for 25 sec, 72°C for 20 sec. Three biological replicates with technical duplicates were performed for the RT-qPCR.

After treatment with 100 μM MeJA, 14-day-old rice plants of WT, OsRIP1-OE line J and line H were cultured in a plant chamber at 28°C with 16 h light: 8 h dark. Plant health development was monitored longitudinally (0, 3, 6, 9, 24 and 48 h) through multispectral imaging analysis, including efficiency of photosystem II (Fv/Fm) (Baker, 2008), chlorophyll index (Chlldx) (Gitelson et al., 2003), modified anthocyanin reflectance index (mARI) (Gitelson et al., 2009) and biomass approximation based on the number of pixels occupied by the plant. At each timepoint, side-view images were captured to provide a more precise view of rice plants and leaves (n = 15 plants, 3 plants/image). At each timepoint 15 plants were used for the side-view images and discarded after the image acquisition. All the images were captured by a custom-build multispectral imaging- and microdispenser platform, equipped with WIWAM system and 6-Mp 16-bit 3CCD top-viewer camera (PhenoVation B.V., Wageningen, The Netherlands). Data processing was performed using the “Data Analysis Software” program (PhenoVation B.V.). Additionally, the phenotypic differences between WT and OsRIP1-OE transgenic plants were analyzed for biomass, shoot length and root length after 48 h of mock treatment or MeJA treatment, respectively.

Frozen shoot samples from WT plants subjected to 100 μM MeJA treatment were crushed in the presence of liquid nitrogen. The powder was homogenized in ice-cold extraction buffer containing 25 mM Tris-HCl, 15 mM MgCl₂, 150 mM NaCl, 0.1% NP-40, 1 mM PMSF, 1 μM E64 and 0.1% benzonase (pH 7.6) in a ratio of 2:1 (2 ml of buffer per gram of plant material). Samples were vortexed for 30 seconds followed by 30 seconds cooling on ice for a total of 10 minutes, followed by incubation on a rotary shaker for 30 minutes. The supernatant was recovered by centrifugation at 14,000 rpm for 20 min at 4°C and used as the protein extracts for the pull-down assays. All steps were performed on ice. Protein content of the extracts was determined using the Bradford method (Bradford, 1976).

Recombinant OsRIP1 was produced in E. coli strain Rosetta (DE3) grown at 14°C for 72 h, and purified as described previously (De Zaeytijd et al., 2019).

Pull-down assays were performed using purified recombinant OsRIP1 as bait and protein extracts from shoots of MeJA-treated WT plants as prey. Briefly, 100 μg total rice protein was supplemented with approximately 54 μg purified OsRIP1, and incubated for 30 min at 4°C. 25 μl of Ni-NTA agarose beads (Qiagen, Hilden, Germany) were equilibrated with binding buffer (mix of the phosphate buffer from OsRIP1 purification and plant protein extraction buffer in a ratio of 8.3:1). The beads were incubated with the mix of protein extracts and OsRIP1 for 30 min at 4°C. After centrifugation, the beads were washed once with the binding buffer containing 50 mM imidazole followed by three washes with trypsin digest buffer (20 mM Tris-HCl, 2 mM CaCl₂, pH 8.0). The beads were resuspended in 150 µl trypsin digest buffer and stored at -20°C prior to LC-MS/MS analysis. All incubations were performed on ice. Ni-NTA beads incubated with plant protein extracts only were used as a control. Four biological replicates were performed in total, one replicate was used for silver staining and Western blot analysis, and the other three replicates were used for LC-MS/MS analysis.

Proteins were separated by SDS-PAGE using 15% acrylamide gels (Laemmli, 1970), followed by visualization with Pierce™ Silver Stain Kit (Thermo Scientific, Waltham, MA, United States). For Western blot analysis proteins were transferred from the acrylamide gel to polyvinylidene fluoride transfer membranes (FluoroTrans^® PVDF, Pall Laboratory, USA). Membranes were blocked in Tris-buffered saline (TBS: 10 mM Tris, 150 mM NaCl, 0.1% (v/v) Triton X-100, pH 7.6) containing 5% (w/v) non-fat milk powder. Subsequently, membranes were incubated with the primary anti-His antibody (1:1000, Thermo Fisher Scientific) for 1 h. Membranes were washed three times with TBS prior to incubation with rabbit anti-mouse IgG secondary antibody labelled with horseradish peroxidase (1:10,000, Dako, Glostrup, Denmark) for another hour. Following two washes with TBS and one wash with 0.1 M Tris buffer (pH 7.6), blots were detected and visualized using 0.025% (w/v) 3,3’-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) containing 0.003% (v/v) hydrogen peroxide.

All proteins were separated from Ni-NTA beads by trypsin digest. After acidification and purification, purified peptides were determined by LC-MS/MS analysis using an Ultimate 3000 RSLC nano LC (Thermo Fisher Scientific, Bremen, Germany) in-line connected to a Q Exactive mass spectrometer (Thermo Fisher Scientific) equipped with a pneuNimbus dual ion source (Phoenix S&T). LC-MS/MS runs were searched using the MaxQuant algorithm (version 1.6.3.4) with mainly default search settings, including a false discovery rate set at 1% on both the peptide and protein level. Spectra were searched against the OsRIP1-6x His sequence and Oryza sativa proteins in the Uniprot database (database release version of March 2021 containing 39,947 protein sequences) (www.uniprot.org). Only proteins with at least one unique or razor peptide were retained leading to the identification of 1,443 proteins. Proteins were quantified by the MaxLFQ algorithm integrated in the MaxQuant software. A minimum ratio count of two unique or razor peptides was required for quantification. Further data analysis was performed with the Perseus software (version 1.6.2.1) after loading the protein groups file from MaxQuant.

Reverse database hits were removed, and replicate samples were grouped. Proteins with less than three valid values in at least one group were removed and missing values were imputed from a normal distribution centered around the detection limit leading to a list of 950 quantified proteins that was used for further data analysis. To compare protein abundance between OsRIP1-treated and OsRIP1-nontreated (CTRL) samples, statistical testing for differences between these two groups was performed, using the package limma. Statistical significance for differential regulation was set at FDR = 0.05 and |log₂FC| = 1, and a volcano plot was also generated. The proteins shown to be differentially abundant between groups were visualized in a heatmap after non-supervised hierarchical clustering of z-scored protein LFQ intensities. The proteomics analyses were performed by the VIB Proteomics Core, Center for Medical Biotechnology, UGent Department of Biomolecular Medicine. Mass spectrometry was performed in triplicates.

Protein-protein docking between OsRIP1 and the proteins identified in the pulldown assays has been performed using ClusPro 2.0 (Kozakov et al., 2017) with standard settings. Protein structure files in.pdb format have been obtained using AlphaFold2 (Jumper et al., 2021) using their respective Uniprot codes ( Table 1 ) and selected based on model quality, with low-quality models being removed from further analysis. Simulations followed steps including rigid-body docking through the fast Fourier transform approach, root-mean square deviation clustering of structures, and refinement of the structures on the representative clusters. Poses were selected based on the most overrepresented clusters.

The representative pose for each complex between OsRIP1 and the target proteins underwent rescoring utilizing the Protein Interaction Z Score Assessment (PIZSA) online server (Roy et al., 2019), an empirical scoring function that assesses the stability of protein assemblies taking into consideration amino acid pair preferences. A distance threshold of 8 Å was applied, and PIZSA scores exceeding 0.8 were deemed indicative of possible binding, with values surpassing 1.0 indicating favorable binding.

Each treatment comprised at least three independent biological replicates. All results were presented as mean ± standard deviation (SD) except for the result of plant health parameters that were presented as mean ± standard error of mean. Data were analyzed by the t-test and one-way analysis of variance (ANOVA) using SPSS 17.0 (SPSS Inc., Chicago, IL). Multiple testing correction was performed with the Duncan correction, considering *p < 0.05, **p < 0.01, ***p < 0.001 as significance. For the analyses of the multispectral proxies, Mock and MeJA treated plants were pairwise compared as per independent sample t-test, implementing a Bonferroni multiple-significance test correction.

Phenotypic analysis on 14-day-old rice plants from two independent transgenic lines overexpressing OsRIP1 (OsRIP1-OE) revealed no visible morphological changes when compared to WT plants ( Figure 1B ). Under normal growth conditions no significant differences in total biomass and shoot length were observed for WT plants and plants from transgenic lines. Although plants from line J revealed a significantly shorter root length compared to plants from line H, no significant differences in root length were observed for line J compared to WT plants ( Figure 1C ).

MeJA exposure for 48h caused a significant inhibition of shoot growth in WT plants and plants from line H, unlike plants from line J. Moreover, plants from line H exhibited significantly longer roots compared to WT plants and line J during both normal growth conditions and MeJA exposure for 48 h ( Figure 1C ).

To assess the effects of exogenously applied MeJA on WT plants and OsRIP1-OE plants of both line J and line H, the multispectral imaging platform was employed to visualize multispectral phenomics from the side view (n = 15 plants, 3 plants/image, Supplementary Materials 1 , Supplementary Figure S7 ). All mock-treated plant groups consistently showed a non-significant increase in shoot biomass compared to the plants treated with MeJA for 48 h ( Figure 2A ; Supplementary Materials 1 , Supplementary Figure S8A ).

The effect of MeJA on the efficiency of photosystem II was evaluated as chlorophyll fluorescence (Fv/Fm; Figure 2B ; Supplementary Materials 1 , Supplementary Figure S8B ). MeJA treatment for 3 h yielded slightly lower Fv/Fm-values in WT plants, while slightly higher Fv/Fm-values were apparent in plants of both line J and line H. MeJA treatment for 24 h resulted in significantly lower Fv/Fm-values in both line J and line H, while WT plants remained unaffected. This showed to be a transient effect since no significant differences were observed between plants of both line J and line H and WT plants after 48 h of MeJA exposure.

The effect of MeJA on the leaf chlorophyll content was evaluated as the chlorophyll index (ChlIdx; Figure 2C ; Supplementary Materials 1 , Supplementary Figure S8C ). After 24 h, MeJA-treated plants from both transgenic lines showed impaired ChlIdx-values compared to the untreated (mock) plants, which was not the case for the WT plants.

Plants treated with MeJA were hallmarked by slightly lower (non-significant) mean mARI-values after 3 h for all three plant groups ( Figure 2D ; Supplementary Materials 1 , Supplementary Figure S8D ). This was a constant trend in WT plants as well as plants from line H up to 24 h. After 48h the opposite effect was observed, MeJA treatments resulted in slightly higher mARI values compared to mock treated plants. For plants from line J, the same trend was observed, except for timepoints 6 h and 9 h.

Transcriptome analysis and GO analysis revealed that the enrichment of “S-linalool synthase activity” (in shoots) and “terpene synthase activity” (in roots) in molecular function was characteristic for OsRIP1-OE plants from line J ( Supplementary Materials 1 , Supplementary Figure S9 , Supplementary Tables S15 , S16 ). Among these genes assigned was a monoterpene linalool encoded by Os02g0121700 with a defensive function in plants ( Supplementary Materials 1 , Supplementary Figure S9 ; Supplementary Table S17 ).

mRNA sequencing was performed for shoot and root samples harvested from WT plants and T4 OsRIP1-OE transgenic plants from line J subjected to MeJA treatment or mock treatment. Plants from line J treated with MeJA for 3 h yielded 215 differentially expressed and down-regulated genes unique for shoots. Among these 199 annotated genes were significantly enriched in GO terms of “chlorophyll biosynthetic process”, “regulation of cellular process” and “response to stimulus” ( Figure 3B1 ; Supplementary Materials 1 , Supplementary Table S2 ). We also identified 125 differentially expressed and up-regulated genes in these shoots ( Figure 3A1 ) but no Gene Ontology (GO) enrichment results were found for the 114 annotated genes.

For 358 annotated genes down-regulated only in shoots of WT plants after MeJA exposure for 3 h (MeJA vs. mock, 3 h), GO enrichment analysis indicated two biological processes, in particular “DNA unwinding involved in DNA replication” and “DNA replication initiation” with the highest p-value ( Figure 3B1 ; Supplementary Materials 1 , Supplementary Table S3 ). When rice plants were subjected to MeJA stress for 24 h, GO terms of photosynthesis-related biological processes were over-represented in 330 down-regulated annotated genes only found in shoots of plants from line J ( Figure 3A2 ). GO terms related to negative regulation of cell cycle were characteristic for the set of 437 down-regulated transcripts unique in WT plants exposed to MeJA for 24 h ( Figure 3B2 ; Supplementary Table S6 ). GO ontology analyses were also performed for the root samples. An enrichment of the biological processes important for N metabolism such as the cellular nitrogen compound metabolic process (GO:0034641) was found in the set of down-regulated genes in roots of WT plants after MeJA treatment for 3 h, while this phenomenon was found in roots of OsRIP1-OE plants from line J at 24 h after MeJA exposure ( Supplementary Materials 1 , Supplementary Figure S6 ; Supplementary Tables S9 – S14 ).

Transcript numbers for genes related to JA signaling ( Figures 4A–D ), GA signaling ( Figures 4E–H ), cytokinin signaling ( Figures 4I–L ), salicylic acid signaling ( Figures 4U–X ), cell cycle ( Figures 4M–P ), and photosynthesis ( Figures 4Q–T ) were analyzed. MeJA treatment for 3 h activated many genes associated with the JA pathway. Among 82 common JA-related genes up-regulated after 3 h of MeJA treatment, 60 differentially expressed genes (DEGs) showed a higher up-regulation in WT plants compared to plants from line J ( Table 2 ; Figure 4A ). 24 up-regulated genes were unique for shoots of WT plants, while there were only 3 up-regulated unique DEGs for shoots of line J ( Table 2 ; Figure 4A ). After MeJA treatment for 24 h, a higher number of down-regulated genes were identified in shoots from line J than in WT plants ( Table 2 ; Figure 4D ), implying that OsRIP1 overexpression caused the delay of activation of the JA signaling in plants from line J compared to WT plants.

In the set of GA-related genes at 3 h after MeJA exposure, more down-regulated genes were identified in shoots from line J than in WT plants ( Table 2 ; Figure 4F ). Compared to plants from line J, 24 h exposure to MeJA led to a higher number of up-regulated genes and less down-regulated genes associated with the GA signaling in WT plants ( Table 2 ; Figures 4G, H ), suggesting that the mRNA transcripts associated with the GA pathway occurred in lower levels in OsRIP1 overexpressing plants than in WT plants.

Compared to plants from line J, more cytokinin-related transcripts were found to be down-regulated in WT plants after 24 h of MeJA supplementation ( Figure 4L ). A similar phenomenon was observed for genes associated with cell cycle ( Figure 4P ). It is noted that 3 h of MeJA exposure caused down-regulation of cell cycle-related genes; the number of DEGs in WT plants was double compared to plants of line J ( Figure 4N ). Among photosynthesis-related DEGs suppressed by 24 h of MeJA treatment, two-thirds of the common down-regulated genes were found to be down-regulated at a higher level in WT plants than in plants of line J ( Figure 4T ). Despite the higher number of upregulated transcripts compared to those downregulated in either line within the salicylic acid (SA) mediated signaling pathway, the total number of transcripts with differential expression was limited (< 30) ( Figures 4U–X ), and most of the SA-mediated transcripts exhibited expression changes with a log2 fold change of less than 1 (data not shown). Additionally, slight differences were observed between the MeJA-induced expression changes in SA-related genes in WT plants and those from line J. Despite the well-established crosstalk between salicylic acid and jasmonate (Peng et al., 2021), in this case, MeJA exposure did not primarily affect the SA mediated signaling pathway in either WT plants or OsRIP1-overexpressing plants from line J.

To validate the mRNA-Seq data, 6 genes including 3 genes for photosynthesis (OsRBCS2, OsRBCS4, OsRBCS5), 2 genes involved in the JA signaling pathway (OsHLH148, OsJAZ12) and 1 gene encoding cytochrome P450 (Cytochrome P450) were selected for RT-qPCR analysis ( Supplementary Materials 1 , Supplementary Table S1 ; Figure 5 ). At 3 h after MeJA treatment, WT plants exhibited 3 more times of OsHLH148 transcript expression than plants from line J ( Figure 5D ). OsbHLH148 transcript levels in shoots of plants from line J were more upregulated after 24 h of MeJA treatment compared to 3 h of MeJA treatment, but still significantly lower than that in WT plants ( Figure 5D ). Similarly, MeJA exposure resulted in less up-regulation of the expression of OsJAZ12 in shoots of plants from line J compared to WT plants at 3 h ( Figure 5E ). 24 h of MeJA treatment stimulated more expression of OsJAZ12 in plants from line J than 3 h of MeJA treatment ( Figure 5E ). MeJA application to WT plants for 3 h or 24 h had no discernible effect on OsbHLH148 or OsJAZ12 transcript levels ( Figures 5D, E ). These RT-qPCR expression profiles validate the mRNA-Seq data.

Protein extracts from shoots of WT plants subjected to MeJA treatment were used as prey in pull-down assays to identify potential interaction partners of OsRIP1. Compared to the control group of proteins from Ni-NTA beads only treated with the plant protein extracts ( Supplementary Figure S10A , lane 2), several proteins with a molecular weight between 17 kDa and 26 kDa appeared in the proteins from the bait samples ( Supplementary Figure S10A , lane 1), and one additional one band of less 10 kDa was clearly present.

The three control samples and three bait samples were analyzed by LC-MS/MS runs. In all six samples 44,086 peptides were identified by LC-MS/MS system and attributed to a total of 1,443 proteins, in which 950 proteins could reliably be quantified. For the statistical analysis, protein intensities in bait samples were compared to those in control samples using a t-test approach with the settings at FDR = 0.05 and |log₂FC| = 1 ( Supplementary Materials 2 ). The Log2 fold change and the statistical significance (-log(p-value)) can be seen on the volcano plot ( Supplementary Figure S10C ), and 12 proteins in total were found to be significantly enriched in the bait samples. Of these 12 proteins, OsRIP1 was abundantly present as expected due to the OsRIP1 addition to the bait samples. Other 11 enriched proteins were considered to represent potential interaction partners for OsRIP1, and their localization as well as defined functions were summarized in Table 1 .

Among these 11 potential interactors, four interactors were known to carry nucleotide-binding/recognition domains, of which tubulin alpha-1 chain (α-tubulin), a GTP-binding protein was associated with cytoskeleton organization. Based on the information of subcellular localization in the Uniprot database, 4 out of 11 putative interactors are localized to the chloroplast, while 7 interactors are predicted to be located in the cytoplasm. Three potential interactors were ribosomal proteins, of which only one (40S ribosomal protein S5) is present in the cytoplasm and 2 others (50S ribosomal protein L27 and 30S ribosomal protein S12) reside in the chloroplast. In addition to two ribosomal proteins from the chloroplast, two more proteins containing a transit peptide were also identified plastid lipid associated protein 2 and photosystem II 10 kDa polypeptide, respectively. The transit peptide is required for their transport across the relevant membranes from their site of synthesis in the cytoplasm. Plastid lipid associated protein 2 belongs to the plastid-lipid associated protein/fibrillin family, while photosystem II 10 kDa polypeptide is known to participate in photosynthesis ( Table 1 ).

According to the GO enrichment analysis, the biological processes over-represented in this subset of 12 proteins, including OsRIP1 and its 11 putative interaction candidates, are related to “translation” (GO:0006412) and “cellular amide metabolic process” (GO:0043603), whereas “mRNA binding” (GO:0003729), “RNA binding” (GO:0003723), “structural molecule activity” (GO:0005198) and “structural constituent of ribosome” (GO:0003735) are enriched in the category molecular function ( Table 1 ).

Protein-protein docking was applied to understand the molecular interactions between OsRIP1 and the putative interaction candidates identified by the pull-down assays ( Table 1 ). Docking revealed a favorable binding of OsRIP1 with photosystem II 10 kDa polypeptide ( Supplementary Materials 1 ; Supplementary Figure S11A ), and 40S ribosomal protein S5 ( Supplementary Figure S11B ). Docking also suggested a binding with α-tubulin ( Supplementary Materials 1 ; Supplementary Figure S11C ), but unstable binding was predicted for the remaining proteins tested.