OsMYB306‐OsRAV11 Regulates Resistance of Rice to Striped Stem Borer by Modulating Serotonin Biosynthesis

ABSTRACT

Striped stem borer (SSB; Chilo suppressalis Walker) is one of the most destructive pests in rice production. Previous studies have demonstrated that SSB infestation induces transcription of OsT5H (tryptamine‐5‐hydroxylase) and biosynthesis of serotonin, a newly recognised phytohormone, and that disruption of serotonin biosynthesis significantly increases SSB resistance. However, the regulatory module modulating serotonin biosynthesis remains to be identified and characterised. Here, we reveal an OsMYB306‐OsRAV11 module that regulates OsT5H transcription and serotonin biosynthesis in response to SSB infestation in rice. OsMYB306 and OsRAV11 can bind to the OsT5H promoter and repress its transcription. In the module, OsRAV11 interacts with OsMYB306 and enhances its inhibitory effect on OsT5H transcription. CRISPR/Cas9‐generated knockout mutants (myb306, rav11 and myb306 rav11) exhibited elevated OsT5H expression, increased serotonin accumulation and reduced SSB resistance. Conversely, OsRAV11 overexpression reduced OsT5H transcription. Our findings establish a transcriptional regulatory framework for the biosynthesis of serotonin in response to SSB infestation. These findings inform the development of new strategies for producing SSB‐resistant rice by genome editing, potentially reducing reliance on chemical pesticides for SSB control.

1. Introduction

Rice ( Oryza sativa L.) is a globally significant staple crop and is the primary energy source for nearly half of the world's population (Thapa and Bhusal 2020). However, rice production is severely threatened by various pathogens and pest insects with global yield losses of up to 30% (Savary et al. 2019). Whilst rice plants have co‐evolved genes and pathways for resistance to some insect pests such as brown plant hopper (BPH) (Du et al. 2009), no germplasm showing sufficient levels of resistance to striped stem borer (SSB, Chilo suppressalis Walker), one of the most destructive pests in rice production (Fahad et al. 2015), has been identified to date. The exception to this is transgenic lines expressing Bt genes (Shu et al. 2000) and gene‐edited plants with mutants of the OsT5H gene (Lu et al. 2018). The OsT5H knockout mutants, on the other hand, although resistant to both BPH and SSB, are not suitable as new commercial varieties due to the presence of yield‐affecting lesion mimics in the leaves and sheaths when grown in the field (Lu et al. 2018; Wang et al. 2023). Therefore, SSB control has so far been heavily reliant on the extensive use of chemical pesticides, driving an urgent need to develop resistant varieties for increased rice production.

The OsT5H gene encodes tryptamine‐5‐hydroxylase, which converts tryptamine to serotonin (5‐hydroxytryptamine, 5‐HT) (Fujiwara et al. 2010). 5‐HT, a pineal hormone in animals, was first reported in plants in 1954 (Erland et al. 2015); it has since been shown to play important roles in plant growth and development, as well as in plant response to various environmental stimuli (Sun et al. 2025). In rice, SSB infestation can significantly increase serotonin accumulation (Ishihara et al. 2008). Our previous studies demonstrated that infestation by both BPH and SSB led to increased OsT5H transcription, and hence increased serotonin biosynthesis and accumulation in rice (Lu et al. 2018). In that same study we revealed that the induced OsT5H transcription was blocked in BPH‐resistant rice varieties, and that the OsT5H knockout exhibited significantly enhanced rice resistance to both BPH and SSB. We further demonstrated that the OsT5H knockout does not disrupt the innate defence response of rice plants to SSB infestation, indicating it is a deficiency of serotonin that enhanced SSB resistance rather than the effect of the OsT5H knockout itself on the plant defence response (Wang et al. 2023). Findings from these studies suggest that rice varieties with enhanced resistance could be developed by disrupting the signalling pathway of SSB/BPH‐induced serotonin biosynthesis, which would keep the basal serotonin biosynthesis sufficient for normal plant growth.

Although numerous studies have shown that serotonin biosynthesis and accumulation can be induced by various biotic and abiotic stresses, its underpinning molecular mechanisms remain mostly elusive in plants (Sun et al. 2025). In cassava, Wei et al. (2018) demonstrated that two cassava apetala2/ethylene response factor (AP2/ERF) gene family members, MeRAV1 and MeRAV2, directly bind to the promoter of T5H and activate its transcription (Wei et al. 2018). They further showed that MeRAV1 and MeRAV2 were targeted by the coat protein of cassava common mosaic virus to subvert the immunity of cassava against the virus (Wei et al. 2024). In rice, Sun et al. (2022) demonstrated that UV‐B stress induces OsbZIP18 transcription, and that OsbZIP18 activates serotonin biosynthesis genes, including tryptophan decarboxylase 1 (OsTDC1), tryptophan decarboxylase 3 (OsTDC3) and OsT5H by directly binding to the ACE‐containing or G‐box cis‐elements in their promoters (Sun et al. 2022). These studies indicate that a set of specific transcriptional factors and partner proteins are responsible for the induction of serotonin biosynthesis by a given stress, such as SSB infestation.

With the aim of developing a strategy for breeding SSB resistant rice varieties by modulating OsT5H transcription and serotonin biosynthesis, this study focused both on the identification of transcription factors that regulate OsT5H transcription in response to SSB infestation and elucidating the underlying mechanisms involved. We first identified an R2R3‐MYB transcription factor, OsMYB306, that could directly bind to the OsT5H promoter, and then identified an OsMYB306‐interacting transcription factor, OsRAV11, that could also bind to the OsT5H promoter. We further revealed an OsMYB306‐OsRAV11 regulatory module that synergically represses OsT5H transcription to modulate serotonin biosynthesis and consequently resistance to SSB in rice.

2. Results

2.1. OsMYB306 Binds to the CAACCA Motif in the OsT5H Promoter

To identify transcription factors that regulate OsT5H transcription, a yeast one‐hybrid (Y1H) library screening was performed using the promoter of OsT5H as bait and a rice cDNA library as prey. More than 100 positive clones were detected on plates with a synthetic dropout (SD) medium without leucine but supplemented with 200 ng/mL Aureobasidin A (AbA). Subsequent sequencing of the positive clones revealed cDNA sequences that were part of a total of 32 genes, nine of which were classified as transcription factors (Table S1). Further Y1H analyses for all these 9 transcription factors demonstrated that only LOC_08g33940 could actually bind to the OsT5H promoter (Figure S1).

To maximise the possibility of identifying potential transcription factors that bind to the OsT5H promoter, we performed an in silico analysis of the OsT5H promoter region using PlantRegMap (http://plantregmap.gao‐lab.org/). The results revealed 48 transcription factors with putative binding affinity to the OsT5H promoter (Table S2). Joint analysis of the transcription factors revealed by PlantRegMap analysis (48) and by Y1H library screening (9) identified LOC_Os08g33940 as the sole shared gene. Hence, we focused on the characterisation of LOC_Os08g33940.

LOC_Os08g33940 has previously been reported to be a member of the MYB or MYB‐like gene family in a genome‐wide classification and expression analysis of MYB transcription factor families in rice (Katiyar et al. 2012). This gene is designated as myb‐related protein 306 in the NCBI (LOC9272734 myb‐related protein 306 [Oryza sativa Japonica Group (Japanese rice)] ‐ Gene ‐ NCBI). For simplicity, we have designated this gene as OsMYB306.

To confirm that OsMYB306 binds to the OsT5H promoter, a series of in vitro and in vivo experiments were performed. First, to identify the potential sequence segments to which OsMYB306 might bind, PlantCARE analysis was performed for the OsT5H promoter, revealing the presence of 9 CAACCA motifs (Figure 1A, blue arrows). Second, to identify the specific OsMYB306 binding sites, several vectors (pHIS2‐p1 ~ −p5) with a 500‐bp segment of the OsT5H promoter (p1 ~ p5, Figure 1A) were constructed. Y1H assays to test their interaction with OsMYB306 demonstrated that only the yeast strain co‐expressing pHIS2‐p1 and AD‐OsMYB306 was able to grow on a TDO medium supplemented with 50 mM 3‐amino‐1,2,4‐triazole (3‐AT), similar to the positive control (pHIS2‐p53 and AD‐53) (Figure 1A). In contrast, yeast strains co‐expressing pHIS2‐p2 ~ −5 and AD‐OsMYB306, as well as those co‐expressing pHIS2‐p1 and AD‐53 (negative control), failed to grow under these same conditions (Figure 1A). These results suggest that OsMYB306 specifically binds to motifs within the p1 segment, which indeed hosts two partially overlapped CAACCA motifs (−47 bp ~ −38 bp) (Figure 1A, red square).

FIGURE 1.

OsMYB306 binds to the OsT5H promoter. (A) Upper: Schematic diagram of the OsT5H promoter. Blue arrows indicate the location of potential MYB binding motifs revealed by PlantCARE analysis. Red squares represents two potential binding sequences predicated by PlantRegMap. Bottom: Yeast one‐hybrid assays showing OsMYB306 binding to the promoter fragments of OsT5H. The basal concentration of 3‐AT was 50 mM. AD‐53 and pHIS2‐p53 were used as a positive control. (B) Interaction of the OsMYB306‐His protein with the OsT5H promoter in electrophoretic mobility shift assays (EMSA). Positive control, EBNA extract; Biotin‐Pr‐EBNA, was used as a probe for positive control. Unlabeled probes were used as competitors. ‘+’ and ‘−’ indicate the presence or absence of proteins or probes in the mixtures. (C) Relative LUC activities in N. benthamiana leaf segments transformed with the ProOsT5H‐LUC together with the indicated effector constructs. Means and standard deviations were calculated from the results of six biological repetitions. Asterisks indicate significant differences of relative Luc/Ren ratio between the indicated effector constructs. (Student's t‐test, **p < 0.01).

The interaction between OsMYB306 and the OsT5H promoter was further investigated through electrophoretic mobility shift assays (EMSA) using a recombinant OsMYB306‐His fusion protein. A distinct DNA‐protein complex was observed when a CAACCA‐containing oligonucleotide was used as a labelled probe. However, complex formation was reduced with increasing amounts of competitor (unlabeled probe) (Figure 1B). Furthermore, no DNA‐protein complex was detected when a mutated probe was used (Figure 1B). These results confirmed that the binding of OsMYB306 to the OsT5H promoter requires the CAACCA sequence.

Further studies were carried out to test whether OsMYB306 regulates OsT5H transcription in vivo using dual‐LUC reporter assays in N. benthamiana leaves. The results demonstrated that luciferase activity in regions co‐transformed with 35S::OsMYB306 and ProOsT5H‐LUC was significantly lower than those co‐transformed with 35S::GFP and ProOsT5H‐LUC (negative control) (Figure 1C). These results demonstrated that OsMYB306 can repress OsT5H transcription in vivo, supporting its role as a transcriptional repressor.

Collectively, the above results demonstrate that OsMYB306 binds to the CAACCA motif in the promoter of OsT5H and negatively regulates its transcription in vivo.

In the absence of information either on LOC_Os08g33940 (OsMYB306) or the protein (OsMYB306) it encodes, a series of experiments was performed to better characterise them. Bioinformatic analysis showed that OsMYB306 belongs to the R2R3‐type MYB family, which is distinguished by the presence of two conserved MYB DNA‐binding domains. Subcellular localisation assays showed that OsMYB306 was localised to the nucleus (Figure S2) in rice protoplasts co‐transfected with 35S::OsMYB306‐GFP or 35S::GFP and NLS‐mCherry plasmids.

2.2. OsMYB306 Knockout Increases OsT5H Transcription and Serotonin Accumulation

To elucidate the biological function of OsMYB306 and its regulatory role in the defence response, we first examined the dynamic expression patterns of OsMYB306 and its downstream target OsT5H in response to SSB infestation. In 2‐month‐old rice plants, OsT5H expression increased significantly at 1 h and reached approximately 4‐fold induction at 24 h post feeding onset (Figure 2A). OsMYB306 expression was also induced by SSB feeding, with transcript levels increasing at 1 h and remaining around 2‐fold at 24 h (Figure 2B).

FIGURE 2.

Knockout of OsMYB306 increases OsT5H transcription and serotonin accumulation. (A and B) RT‐qPCR analysis of OsT5H (A) and OsMYB306 (B) expression in 2‐month‐old wild‐type rice plants at 1 h, 8 h and 24 h after the onset of SSB feeding. The control group (CK) without feeding was sampled at the same time points. The expression levels of each gene, normalised to Actin expression, are presented relative to the CK, which is set to 1. Data are shown as mean ± SD of three biological replicates. Five plants in an individual block represent one replicate. Different letters indicate significant difference at p < 0.05 level (Student's t‐test). (C) CRISPR/Cas9‐mediated targeted mutagenesis of OsMYB306. Upper, a schematic diagram of OsMYB306, exons being represented by black boxes and introns by line; the CRISPR/Cas9 target sites are indicated by arrows. Bottom, nucleotide sequences flanking the PAM site (nucleotides underlined) in the two OsMYB306 knockout lines myb306‐1 and myb306‐2, and the wild‐type (WT) plants. (D) RT‐qPCR analysis of OsT5H in 2‐week‐old seedlings of wild‐type, myb306‐1 and myb306‐2. Data are shown as mean ± SD of three biological replicates. Asterisk indicates significant difference between myb306 and wild‐type (Student's t‐test, *p < 0.05, **p < 0.01). (E) RT‐qPCR analysis of OsT5H in 4‐week‐old wild‐type, myb306‐1 and myb306‐2. Plants in the treatment group were subjected to mechanical damage (MD) using a hole puncher, followed by a 24‐h incubation period before being sampled alongside the control group (CK). Data are shown as mean ± SD of three biological replicates. Different letters indicate significant difference at p < 0.05 level (Student's t‐test). (F) 5‐HT levels in rice plants as treated in (C). The quantification was performed using high‐performance liquid chromatography (HPLC). Data are shown as mean ± SD of three biological replicates. Different letters indicate significant difference at p < 0.05 level (Student's t‐test).

To further investigate the function of OsMYB306 on OsT5H transcription and also to elucidate its role in serotonin biosynthesis in rice plants, two OsMYB306 knockout mutants were generated by CRISPR/Cas9‐mediated target mutagenesis (Figure 2C). Sequencing of the targeted genomic regions showed that myb306‐1 and myb306‐2 contained an adenine (A) deletion and a thymine (T) insertion, respectively. Both mutations resulted in frameshifts and truncated proteins (Figure S3). Given that OsT5H transcription is sensitive to the growing conditions of plants, particularly day and night (Wei et al. 2016), OsT5H mRNA abundance was measured at 3 time points across a 24‐h period in 2‐week‐old seedlings of the wild‐type (WT) parental cultivar Jiahua No. 1 and its two OsMYB306 knockout mutants. The results showed that OsT5H mRNA was significantly more abundant in both myb306‐1 and myb306‐2 than in the WT at all time points (Figure 2D), supporting the LUC/REN observation that OsMYB306 acts as a transcriptional repressor of OsT5H.

When SSB larvae bite rice plants, it causes mechanical damage (MD) and delivers salivary effectors into plants. To examine the effects of MD on OsT5H transcription, we investigated OsT5H mRNA levels in WT rice plants either treated with MD, SSB mock feeding (MD + SSB saliva), or directly infested with SSB. The results showed that all three treatments significantly increased OsT5H transcription compared to the control plants. However, there were no differences between these treatments (Figure S4). This finding supports the use of MD as an alternative to SSB feeding in subsequent experiments related to transcriptional responses.

To determine the mutational effects of OsMYB306, we first examined OsT5H transcription in WT and mutant plants. MD treatment induced OsT5H expression in both mutants compared with WT, but caused a greater increase in myb306‐1 and myb306‐2 than that in the WT (Figure 2E).

To determine the effects of OsT5H mutation on serotonin biosynthesis, changes in serotonin content in the WT and mutant rice plants were measured using high‐performance liquid chromatography (HPLC). Consistent with the elevated OsT5H mRNA levels, the serotonin content was significantly higher in the two mutants than in the WT, particularly in those subjected to MD (Figure 2F). The serotonin content was 3.23 μg/g and 2.96 μg/g in myb306‐1 and myb306‐2, respectively, significantly greater than that in the WT plants (1.96 μg/g) 24 h post MD.

The findings that the transcription of OsT5H and the serotonin content were significantly greater in myb306‐1 and myb306‐2 than in the WT, either in the presence or absence of MD, are consistent with the dual‐LUC reporter assays, which suggested that OsMYB306 functions as a transcriptional repressor of OsT5H (Figure 1C).

2.3. OsMYB306 Interacts With the Transcription Repressor OsRAV11

To elucidate the in‐depth mechanism underlying OsMYB306‐mediated regulation of OsT5H during SSB infestation in rice, we performed an immunoprecipitation–mass spectrometry (IP‐MS) analysis using protein extracts from rice tissues, including those collected 24 h after mechanical damage and SSB infestation. A total of 652 proteins were identified following the exclusion of results from the control group. Since our aim was to identify potential transcriptional regulators that might interact with OsMYB306, we focused on 16 proteins annotated as transcription factors (Table S3).

We hypothesised that potential OsMYB306‐interacting transcription factors would also respond to SSB damage, hence we analysed the RNA‐seq data of rice plants before and after SSB infestation (Wang et al. 2023). The results indicated that only LOC_Os01g49830 was responsive (Figure S5), with transcript levels being significantly greater in SSB infested plants (277, 207, 125 FPKM, on average 1, 3, 8 h post infestation) than in non‐infested plants (20, 15, 12 FPKM, on average 1, 3, 8 h post infestation).

LOC_Os01g49830 is annotated to encode a RAV (RELATED TO ABI3 AND VP1) family protein, OsRAV11. RAV proteins contain two distinct DNA‐binding domains: an APETALA2 (AP2) domain at the N‐terminus and a B3 domain at the C‐terminus. Previous studies have identified several RAV family transcription factors in Arabidopsis, including AtRAV1 and AtRAV2, which exhibited transcriptional repressive activity (Ikeda and Ohme‐Takagi 2009). Further experiments revealed that this repressive function is mediated by a conserved short peptide motif, R/KLFGV (Ikeda and Ohme‐Takagi 2009). Through homology alignment, we identified OsRAV11 in rice as a homologue of AtRAV1 (AT1G13260) and confirmed the presence of a conserved repression domain RLFGV at the C‐terminal region (Figure S6). In rice, OsRAV11 has been demonstrated to play a role in reproductive development and its knock‐out resulted in elongated carpels (Osnato et al. 2020). In this study, OsRAV11 was identified as a candidate protein interacting with OsMYB306 based on IP‐MS results. Subcellular localisation assays showed that OsRAV11‐GFP accumulated in the nucleus (Figure S7), consistent with its predicted role as a transcription factor.

The interaction of OsRAV11 with OsMYB306 was confirmed in vitro by one‐to‐one Y2H assays. As strong self‐activation was observed when full‐length OsMYB306 was used, several BD constructs with truncated forms of OsMYB306 were generated (Figure 3A). OsMYB306 contains two MYB‐DNA binding domains; however, BD constructs with either one of the two domains did not show any self‐activation, and neither did they interact with OsRAV11 (Figure 3A, column B, C). In contrast, a fragment (286–310 aa) at the C‐terminal end appeared to be crucial for OsMYB306 self‐activation, whilst a different fragment near the C‐terminal region (262–286 aa) was essential for its interaction with OsRAV11(Figure 3A).

FIGURE 3.

OsMYB306 interacts with OsRAV11. (A) Yeast two‐hybrid assay evaluating the interaction between OsRAV11 and various OsMYB306 truncated proteins. Schematic diagrams of OsMYB306 truncated proteins (A–G) that were fused to BD (empty vector). The full‐length OsMYB306 protein contains two Myb_DNA‐binding domains, which are denoted by red squares in the diagram. (B) BiFC assay of the interaction between OsMYB306 and OsRAV11 in N. benthamiana leaves. DAPI, fluorescence of 4′,6‐diamino‐2‐phenylindole; Merge, merged EYFP, DAPI and bright field images. (C) LCI assay of the interaction between OsMYB306 and OsRAV11. The indicated fusion pairs were co‐expressed in N. benthamiana leaves. (D) The interaction between OsMYB306‐4HA and OsRAV11‐3FLAG shown by Co‐IP in rice protoplasts. Total proteins were extracted and immunoprecipitated by anti‐FLAG magnetic beads. The input and immunoprecipitated proteins were detected by anti‐FLAG or anti‐HA antibody.

To further characterise the interaction between OsMYB306 and OsRAV11, a series of complementary studies were performed. Bimolecular fluorescence complementation (BiFC) assays in N. benthamiana leaves demonstrated their interaction in the nucleus in vivo (Figure 3B). Luciferase complementation imaging (LCI) analysis showed strong luciferase signals when they were co‐expressed (OsMYB306‐nLUC and cLUC‐OsRAV11) (Figure 3C). This interaction was further confirmed by swapping the luciferase fusions between the two proteins in tobacco leaves. Furthermore, we validated the interaction between OsMYB306 and OsRAV11 in rice protoplasts using co‐immunoprecipitation (Co‐IP). Expression vectors for Ubi::OsMYB306‐4HA and Ubi::OsRAV11‐3FLAG, as well as the control vector Ubi:3FLAG, were constructed and used for transformation into rice protoplasts. Tagged proteins were enriched using anti‐Flag magnetic beads for Western blot analysis. The results showed the co‐precipitation of OsMYB306 with OsRAV11 in protoplasts transfected with Ubi::OsMYB306‐4HA and Ubi::OsRAV11‐3FLAG, but not in those transfected with Ubi::OsMYB306‐4HA and Ubi::3FLAG (Figure 3D). Collectively, these data clearly demonstrate that OsMYB306 physically interacts with OsRAV11 in rice plants.

2.4. OsRAV11 Binds to the OsT5H Promoter and Cooperatively Represses Its Transcription With OsMYB306

Members of the RAV family possess highly conserved AP2 or B3 DNA binding domains (Chen et al. 2021). A previous study demonstrated that in Arabidopsis, the AP2 and B3‐like domains recognise the CAACA and CACCTG motifs, respectively (Kagaya et al. 1999). To explore the relationship between OsRAV11 and OsT5H, we analysed the cis‐regulatory elements in the OsT5H promoter and identified five CAACA motifs, but no CACCTG motifs. To identify which CAACA motif OsRAV11 binds to, ChIP‐qPCR assays on protoplasts transfected with the 35S::OsRAV11‐4HA construct were performed. DNA fragments that bound to OsRAV11 were immunoprecipitated using an anti‐HA antibody and used for qRT‐PCR analysis with 5 pairs of PCR primers designed for the amplification of DNA fragments covering the CAACA motif (F1 ~ F5, Figure 4A). Among them, F3 and F4, which are located in the OsT5H promoter (1718 bp −1585 bp; 881 bp −739 bp, respectively), showed significant enrichment (Figure 4A). To further identify which CAACA motifs OsRAV11 binds to, Y1H assays with AD‐OsRAV11 as prey and oligonucleotides of different CAACA motif‐containing segments (portion of F1‐F6, Figure 4A) as bait were carried out. To enhance the effect of these Y1H assays, a 20‐bp fragment containing the CAACA motif was synthesised for each site and cloned into a pHIS2 vector with 3 copies of the 20‐bp fragment in tandem; the vectors were designated as pHIS2‐f1 to ‐f6, respectively. Yeast strains co‐expressing pHIS2‐f1, −3 and ‐f4 and AD‐OsRAV11 were able to grow on TDO + 3‐AT medium, but those co‐expressing pHIS2‐f2, −5 and ‐f6 and AD‐OsRAV11 were not (Figure 4B). These results, together with the ChIP‐qPCR results, suggest that OsRAV11 binds to the OsT5H promoter through the CAACA motif regions between 1718 bp to 1585 bp and 881 bp to 739 bp.

FIGURE 4.

OsRAV11 Binds to the OsT5H promoter and cooperatively represses its transcription with OsMYB306. (A) ChIP‐qPCR analysis of OsRAV11 binding to the promoter of OsT5H. Upper, a schematic diagram of OsT5H and its promoter. The arrows indicate the location of CAACA motifs, and F1‐F6 are six fragments in ChIP‐PCR. Bottom, ChIP‐PCR results, data with different letters are significantly different at p < 0.05 level. Error bars indicate SD. (B) Yeast one‐hybrid assays showing OsRAV11 binding to the promoter fragments of OsT5H. The basal concentration of 3‐AT was 50 mM. AD‐53 and pHIS2‐p53 were used as a positive control. (C) RT‐qPCR analysis of OsRAV11 in 2‐month‐old wild‐type plants at 1, 8 and 24 h after the onset of SSB feeding. The CK without feeding was sampled at the same time points. Data are shown as mean ± SD of three biological replicates. Five plants in an individual block represent one replicate. Different letters indicate significant difference at p < 0.05 level (Student's t‐test). (D) CRISPR/Cas9‐mediated targeted mutagenesis of OsRAV11. (E) RT‐qPCR analysis of OsT5H in 4‐week‐old wild‐type, rav11‐1 and rav11‐2 plants. Plants in the treatment group were subjected to mechanical damage using a hole puncher, followed by a 24‐h incubation period before being sampled alongside the control group. Data are shown as mean ± SD of three biological replicates. Different letters indicate significant difference at p < 0.05 level (Student's t‐test). (F and H) RT‐qPCR analysis of OsRAV11 and OsT5H in independent 2‐week‐old T2 Ubi:OsRAV11‐3FLAG Lines. The levels of OsRAV11 and OsT5H expression, normalised to Actin expression, are shown relative to the level in wild‐type plants, which was set to 1. Data are shown as mean ± SD of three biological replicates. Asterisks indicate significant differences between wild‐type and Ubi:OsRAV11‐3FLAG Plants (Student's t‐test, **p < 0.01). (G) Validation of the OsRAV11‐3Flag fusion protein using an Anti‐Flag antibody. (I) Relative LUC activities of the ProOsT5H‐LUC reporters co‐transformed with the indicated effector constructs. Means and standard deviations were calculated from the results of six biological replicates. Different letters indicate significant difference at p < 0.05 level (Student's t‐test). Error bars indicate SD.

To investigate the role of OsRAV11 on OsT5H transcription in rice plants, two transgene‐free, homozygous Osrav11 knock‐out mutant lines (rav11‐1, rav11‐2) were generated by CRISPR/Cas9‐mediated target mutagenesis (Figure 4D; Figure S8). qRT‐PCR analysis showed that the expression levels of OsT5H in rav11‐1 and rav11‐2 were comparable to those in the wild‐type plants under normal conditions (Figure 4E). However, after 24 h of MD, OsT5H transcript abundance in both mutant lines became significantly higher than that in the wild‐type (Figure 4E).

Overexpression lines of OsRAV11 were produced by expressing Ubi::OsRAV11‐3Flag in Jiahua No. 1. Of the 22 lines generated, three lines (#5, #6 and #21) exhibited a moderate increase in OsRAV11 transcription, with up to 4‐fold increase of transcripts (Figure 4F), and corresponding increase in expression of OsRAV11‐3Flag fusion protein (Figure 4G), compared to the wild‐type. As expected, the OsT5H transcript abundance was significantly reduced in all these lines compared to the wild‐type plants (Figure 4H).

To further validate the regulatory effects of OsMYB306 and OsRAV11 on OsT5H transcription, dual‐luciferase reporter assays were performed in N. benthamiana leaves. Co‐expression of ProOsT5H‐LUC with 35S::OsMYB306 or 35S::OsRAV11 markedly reduced luciferase activity compared with the 35S::GFP control (Figure 4I). When ProOsT5H‐LUC was co‐expressed with both 35S::OsMYB306 and 35S::OsRAV11, the LUC/REN ratio decreased to the lowest level (Figure 4I), indicating that OsMYB306 and OsRAV11 act synergistically to repress OsT5H promoter activity.

Taken together, these findings demonstrate that OsRAV11 directly binds to the OsT5H promoter and acts together with OsMYB306 to repress its transcription. This cooperative regulation may play an important role in mediating serotonin‐associated defence responses.

2.5. Analysis of Expression Patterns of OsT5H , OsMYB306 and OsRAV11

To better understand the mechanism by which OsMYB306 and OsRAV11 regulate OsT5H transcription in rice, we analysed their spatiotemporal expression patterns using common sets of plant materials.

We first analysed their expression in various tissues of plants at the flowering stage. These results showed that OsT5H was expressed strongly in the leaf and root but only weakly in the stem, panicle and floret (Figure 5A). In contrast, OsMYB306 was predominantly expressed in the stem and panicle, with lower levels in leaf and root (Figure 5B). OsRAV11 was expressed predominantly in the leaf and mildly in the stem, but barely in root, panicle and floret tissues (Figure 5C).

FIGURE 5.

Expression patterns of OsT5H, OsMYB306 and OsRAV11 in different tissues and developmental stages. (A–C) RT‐qPCR analysis of OsT5H (A), OsMYB306 (B) and OsRAV11 (C) expression in different tissues of wild‐type rice plants at the flowering stage. Data are shown as mean ± SD of three biological replicates and the expression level in the stem was set to 1. (D–F) RT‐qPCR analysis of OsT5H (D), OsMYB306 (E) and OsRAV11 (F) expression in leaves at the seedling, jointing and flowering stages in wild‐type rice plants. Data are shown as mean ± SD of three biological replicates and the expression level at the seedling stage was set to 1. (G, H) Relative transcript levels of OsT5H (G) and OsMYB306 (H) in 2‐week‐old wild‐type and rav11 mutant lines (rav11‐1, rav11‐2) under normal conditions. Data are shown as mean ± SD of three biological replicates (Student's t‐test, *p < 0.05, **p < 0.01). (I) Relative transcript levels of OsRAV11 in 2‐week‐old wild‐type and myb306 mutant lines (myb306‐1, myb306‐2) under normal conditions. Data are shown as mean ± SD of three biological replicates (Student's t‐test, *p < 0.05, **p < 0.01).

We next examined their transcript abundance in leaves of wild‐type rice plants at different developmental stages. All three genes were expressed from the seedling to flowering stages but displayed distinct temporal patterns. OsT5H expression increased from the seedling to the jointing stage and then declined at the flowering stage (Figure 5D). OsRAV11 showed a similar pattern, but with the highest transcript level at the jointing stage (Figure 5F). In contrast, OsMYB306 expression gradually declined over time, with the highest level at the seedling stage and the lowest at flowering (Figure 5E).

We already showed that OsT5H expression was significantly upregulated in 2‐week‐old and 4‐week‐old myb306 seedlings as compared to the WT without MD (Figure 2D,E), whereas no change was observed in the rav11 mutants (Figures 4E and 5G). When treated with MD, OsT5H transcription was significantly increased in both myb306 and rav11 mutants as compared to the WT. Since OsMYB306 and OsRAV11 act cooperatively to repress OsT5H, we further examined the reciprocal effect of mutations on their own expression. The transcript level of OsMYB306 was significantly elevated in both rav11‐1 and rav11‐2 (Figure 5H), while that of OsRAV11 remained unchanged in the myb306 lines (Figure 5I), as compared to the WT.

We further examined the relative rapidness of transcriptional response of these three genes to mechanical damage. When the stems of flowering plants were punched, all three genes showed significantly increased transcription within 60 min (Figure S9). Among them, OsRAV11 showed the quickest response with a significant increase of transcripts within 20 min, followed by OsT5H and OsMYB306. OsRAV11 also showed the greatest increase of transcript abundance change (over 4‐fold). The transcript abundance remained mostly stable 90 min post treatment.

2.6. Knockout of OsMYB306 and OsRAV11 Reduced Resistance of Rice to SSB

The above results clearly demonstrate that knock‐out of either OsMYB306 or OsRAV11 increases OsT5H transcription after MD treatment, resulting in increased serotonin accumulation. Serotonin is essential for SSB larval growth and development (Wang et al. 2023), thus we hypothesised that the OsRAV11 and OsT5H mutations would affect SSB growth, development and survival. To evaluate their combined effect, a double mutant line (designated myb306‐2 rav11‐1) was developed by knocking out OsRAV11 in the myb306‐2 line. Insect bioassays were performed by feeding newly hatched SSB larvae rice stems with sheath cut from plants at the flowering stage.

First, we observed that SBB larvae fed on mutant lines (myb306‐2, rav11‐1 and myb306‐2 rav11‐1) grew much better than on the WT and were notably larger 10 days post infestation (DPI) (Figure 6A). Consequently, the body weight of larvae fed on mutant lines was significantly greater than those fed on the WT, with mean body weights (±SD) of 6.26 ± 3.18 mg (10 DPI) to 29.16 ± 16.31 mg (20 DPI) for myb306‐2, 7.99 ± 3.48 mg (10 DPI) to 37.97 ± 12.27 mg (20 DPI) for rav11‐1 and 6.50 ± 2.88 mg (10 DPI) to 34.53 ± 11.56 mg (20 DPI) for myb306‐2 rav11‐1, compared with 4.55 ± 3.05 mg (10 DPI) to 21.86 ± 8.31 mg (20 DPI) for the WT (Figure 6B).

FIGURE 6.

Knockout of OsMYB306 and OsRAV11 reduced resistance of rice to SSB. (A) Performance of SSB larvae fed WT, myb306‐2, rav11‐1 or myb306‐2 rav11‐1 plants at 10, 15 and 20 days post‐infestation. DPI, days post infestation. Scale bars, 1 cm. (B, C) Weight and mortality of SSB larvae fed WT, myb306‐2, rav11‐1 or myb306‐2 rav11‐1 plants at 10, 15 and 20 days post‐infestation; (B) are shown as boxplots; and (C) are represented by mean ± SEM of 10 biological replicates. Different letters indicate significant difference at p < 0.05 level (Student's t‐test). DPI, days post infestation.

Mortality was also recorded over the same time period. The results showed that the mortality rate of larvae fed WT plants increased from 33% (10 DPI) to 57% (20 DPI). This rate was significantly higher than those for larvae fed myb306‐2 (25% to 37%), rav11‐1 (12% to 20%), or myb306‐2 rav11‐1 (13% to 31%) over the same period (Figure 6C). Notably, no significant differences in mortality were observed among the larvae feeding on the three different mutant lines. Furthermore, the increase in larval growth observed on the mutant lines resulted in an increased rate of development, with some of the larvae having already entered the pupal stage 20 days post hatching, compared to none on the WT rice (Figure S10).

The transcript levels of OsT5H and 5‐HT contents were further quantified in WT and mutant plants before and after 24 h of SSB feeding. OsT5H transcription and 5‐HT accumulation were significantly induced by SSB attack in all genotypes, but their levels were markedly higher in the mutants than in the WT after infestation (Figure S11A,B). The trends observed in rav11‐1 and myb306‐2 rav11‐1 were similar and slightly higher than in myb306‐2, suggesting that OsRAV11 may play a predominant role in the cooperative repression module controlling OsT5H transcription. These results further confirm that disruption of OsMYB306 or OsRAV11 derepresses OsT5H transcription, leading to elevated serotonin production.

Together, these results demonstrate that, consistent with the increased serotonin biosynthesis, the knockout of OsMYB306 and OsRAV11, alone or combined, significantly reduced rice resistance to SSB, leading to improved larval growth, survival and development.

2.7. Natural Variation and Haplotype Analysis of OsT5H , OsMYB306 and OsRAV11

To identify potential functional variants that might be useful in breeding SSB‐resistant rice, we analysed natural variation in the coding regions of OsMYB306 and OsRAV11, as well as the promoter and coding sequences of OsT5H in the 3 k rice accessions currently available (Mansueto et al. 2016). No single nucleotide polymorphism (SNP) was detected in OsMYB306 and only one synonymous SNP (G/A at position 1125 bp, amino acid T375T) was found in OsRAV11.

In comparison, there was relatively rich diversity of OsT5H across the accessions. In its coding sequence, there was a total of 11 SNPs, including 2 non‐synonymous SNPs (M9V and S32L) and 9 synonymous SNPs. There were also several SNPs in the promoter region; however, none of them are in the binding site of OsMYB306 (−47 bp ~ −38 bp) and OsRAV11 (−1616 bp ~ −1612 bp, −809 bp ~ −805 bp) (Figure S12A).

Based on these SNPs in the OsT5H coding region, the 3 k rice population can be classified into 7 major haplotypes, with japonica accessions predominantly carrying Hap1 and indica varieties displaying higher haplotype diversity (Figure S12B). Similarly, these rice accessions can be divided into 3 major haplotypes based on the SNPs in the promoter region.

3. Discussion

With the aim of elucidating the regulatory mechanisms responsible for the induction of serotonin biosynthesis by SSB infestation in rice, this study identified a regulatory module that represses, rather than activates, serotonin biosynthesis. This module consists of two interacting transcription factors, OsMYB306 and OsRAV11, both of which can directly bind to the OsT5H promoter, but at different sites. Further, the interaction between OsMYB306 and OsRAV11 cooperatively enhances the repression of OsT5H transcription, indicating that the two proteins function as a synergistic repressor complex. These results not only shed light on how to increase the resistance of rice to SSB via regulation of this module, but also on the regulation of the biosynthesis of serotonin, a phytohormone of emerging importance in plants.

Both OsMYB306 and OsRAV11 are responsive to SSB infestation, with their encoded proteins binding to the OsT5H promoter. This, together with the findings that OsMYB306 and OsRAV11 are interacting transcriptional factors colocalised in the nucleus, strongly suggests they function as a regulatory module in serotonin biosynthesis.

The co‐existence of transcription factors that synergistically regulate gene expression is a conserved strategy in response to variable biotic and abiotic challenges in plants. In rice, MYB22 interacts with the corepressor TOPLESS through its EAR motif and recruits the histone deacetylase HDAC1 to form a MYB22–TOPLESS–HDAC1 complex. This tripartite complex functions as a transcriptional repressor that directly binds to the promoter of F3′H, a flavonoid biosynthesis gene negatively associated with rice resistance to the brown planthopper (BPH), thereby repressing its expression and enhancing host defence (Sun et al. 2023). Similarly, under drought stress, OsMYBR57, an MYB‐related transcription factor, interacts with the homeodomain protein OsHB22 to coordinately regulate the expression of drought‐related OsbZIPs (Yang et al. 2022). This cooperation ensures precise modulation of stress‐responsive pathways by integrating distinct transcription factor families and their subcellular dynamics.

In our study, SSB infestation activated the transcription of both OsMYB306 and OsRAV11, as well as OsT5H. We demonstrated that OsMYB306 and OsRAV11 could jointly repress OsT5H expression in vitro and in vivo. Their suppressive effect on OsT5H expression was further supported by the fact that, under SSB infestation, OsT5H transcript levels were significantly higher in both the rav11 and myb306 mutants. The results from spatiotemporal expression analyses also supported the above conclusion: The high expression of OsMYB306 and OsRAV11 in stems coincides with the low expression of OsT5H. Since SSB mainly feeds on stems in rice, this suppressive OsMYB306‐OsRAV11 module on OsT5H expression is likely to play an important role in SSB resistance. Expression of OsMYB306 and OsRAV11 was consistently low in roots, where that of OsT5H was moderately high. The finding that there was also a high level of OsT5H transcripts in leaves, where OsRAV11 is highly expressed, but OsMYB306 is not, further suggests that together OsMYB306 and OsRAV11 cause greater suppression on OsT5H than either alone.

The knockout effects of OsMYB306 or OsRAV11 on OsT5H expression were not exactly the same as their differential expression as observed in different tissues. In plants of ~2‐month‐old, OsT5H expression was significantly higher in SSB infested rav11 mutants than in myb306 mutants (Figure S11A). This finding was not surprising since the expression of OsMYB306 had already declined in these older plants while OsRAV11 remained at a high level (Figure 5E), therefore, the mutational effect of the former was expected to be lower than that of the latter. Similarly, it is not unexpected that there was no significant difference in OsT5H expression between the double mutant myb306‐2 rav11‐1 and single rav11 mutants (Figure S11A). The mutational effect on OsT5H expression observed above was ultimately reflected by the concentration of serotonin in rice stem (Figure S11B), i.e., significantly greater serotonin concentrations in both single and double mutant lines of OsMYB306 or OsRAV11 than in the WT.

We have previously demonstrated that suppression of serotonin synthesis increases SSB resistance in rice (Lu et al. 2018) and supplementation of serotonin in the diet improves larval growth and survival (Wang et al. 2023). Here, we further demonstrate that an increase of endogenous serotonin in the OsMYB306 or OsRAV11 knockout mutant lines improved SSB larval growth (Figure 6A), with significantly increased body weight (Figure 6B) and survival rate (Figure 6C). These results demonstrate a strong correlation between endogenous serotonin levels and SSB resistance in rice. Generation and analysis of double mutants of OsT5H and OsMYB306/OsRAV11 may reveal if OsMYB306 or OsRAV11 modulates SSB resistance through mechanisms other than serotonin biosynthesis.

A logical next step is to test OsMYB306 and OsRAV11 overexpression lines to explore new strategies for SSB resistance breeding. However, the growth of OsRAV11 overexpression lines was severely impaired (Figure S13) and their fertility was very low. This finding is not unexpected as OsRAV11 is a transcriptional factor that, when constitutively overexpressed, often causes pleiotropic negative effects on plant growth. Whether OsMYB306 overexpression lines exhibit better performance remains to be investigated.

Rice has rich genetic diversity (Nachimuthu et al. 2015). Therefore, another rational way is to explore natural variations for SSB resistance improvement. However, we did not identify any functional diversity of OsMYB306 and OsRAV11 based on their coding sequences. We also did not find nucleotide variations in the OsMYB306/OsRAV11 binding sites in the OsT5H promoter (Figure S12A). Again, this is not surprising as previous screening of rice genetic collections did not identify any accessions that showed resistance to SSB (Xiang et al. 2023).

As discussed above, the transcription of OsT5H and consequently the serotonin level was significantly increased in response to SSB infestation, even though both OsMYB306 and OsRAV11 are functioning in the WT plants, which strongly suggests there are other transcriptional factors that induce OsT5H expression, as a response to SSB attack. Therefore, further studies are required to identify such regulators for SSB resistance breeding.

In the arms race between pest insects and their host plants, there are often trade‐offs between growth and defence in plants. In this study, the OsRAV11 overexpression lines had significantly reduced OsT5H transcription (Figure 4H) hence are expected to have increased SSB resistance. We observed severe growth defects in these OsRAV11 overexpression lines (Figure S13); thus, those defects could be a type of trade‐off that negatively affects rice plant growth. However, there seem to be other agonistic transcriptional factors counteracting the effect of OsMYB306‐OsRAV11, hence no clear trade‐off impact was identified in the wild‐type plants.

Because SSB feeding involves both physical (mechanical damage) and biochemical effects (delivery of saliva elicitors) on rice plants, people would like to know whether OsMYB306‐OsRAV11 is activated by the physical or chemical effect, or both. We observed that mechanical damage alone had the same effect on OsT5H transcription as SSB feeding and mechanical damage plus saliva treatment (Figure S4), and simple mechanical damage increased the transcription of both OsMYB306 and OsRAV11 (Figure S9). A simple understanding of these results would be that physical damage alone could activate the OsMYB306‐OsRAV11 module. Because brown planthopper (BPH) feeding also exerts physical and biochemical effects on rice plants and induces OsT5H transcription and serotonin biosynthesis (Lu et al. 2018), we attempted to retrieve public RNA‐seq data, with the aim to examine the response of OsMYB306 and OsRAV11 to BPH feeding. However, we failed to identify such data; therefore, further studies are needed to examine whether the OsMYB306‐OsRAV11 module is an SSB responsive model or it is a general model responding to physical damage.

In summary, our present study revealed an SSB‐responsive OsMYB306‐OsRAV11 module that represses the transcription of OsT5H and thus plays a positive role in SSB resistance in rice. No functional genetic diversity of this module has been identified in the 3 k collection of rice accessions available to date. Future studies are needed to identify SSB responsive positive regulators of OsT5H expression for the development of SSB resistance breeding strategies.

4. Experimental Procedures

4.1. Plant Materials and Growth Conditions

The japonica rice ( Oryza sativa L.) cultivar ‘Jiahua #1’ was used as the wild‐type material in this study. The knockout mutants myb306‐1, myb306‐2, rav11‐1, rav11‐2 as well as myb306 rav11 double mutants were produced using CRISPR/Cas9‐mediated target mutagenesis. They were developed using two distinct single‐guide RNAs (sgRNAs) to specifically target the first exon of OsMYB306 (LOC_Os08g33940) and OsRAV11 (LOC_Os01g49830), respectively (Figures 2C and 5D). For the development of OsRAV11 overexpressing lines, the full‐length coding sequence of OsRAV11 was PCR‐amplified and cloned into pENTER‐3FLAG entry vector. Error‐free clones were subsequently introduced into the destination vector pHGW to produce expression vectors by LR recombination reactions (Gateway LR Clonase II, Invitrogen). Transgenic plants were produced via Agrobacterium‐mediated transformation (Hiei et al. 1994) of calli induced mature embryos. The primer sequences used to generate the constructs are listed in Table S4.

Field‐grown rice plants were raised under standard paddy conditions in Sanya (Hainan Province, China). For in‐house studies, rice plants were grown hydroponically in Yoshida rice nutrient solution (Yoshida et al. 1976) under controlled environmental conditions in a growth chamber (14 h light/10 h dark, 28°C ± 1°C), with a relative humidity of ~70% ± 10%.

4.2. Yeast One‐Hybrid (Y1H) Assay

Y1H assays were performed following the Matchmaker One‐hybrid System (Clontech). For library screening, the promoter sequence of OsT5H (2000 bp) was cloned into the pAbAi vector as the bait (pAbAi‐OsT5H) and a rice cDNA library was used as the prey. The constructed pAbAi‐OsT5H plasmid was linearised by restriction enzyme digestion and integrated into the genome of the Y1HGold yeast strain by homologous recombination to generate a bait‐specific reporter strain Y1HGold (pAbAi‐OsT5H). The optimal concentration of Aureobasidin A (AbA) for the bait strain was determined to be 200 ng/mL, which effectively suppressed background growth. The rice cDNA library was transformed into Y1HGold (pAbAi‐OsT5H) following the Matchmaker User Manual. Positive clones were selected on synthetic dropout (SD) medium lacking leucine but supplemented with 200 ng/mL AbA. The cDNA inserts from positive clones were sequenced to identify the corresponding genes. For pairwise interaction validation, different OsT5H promoter fragments were inserted into the reporter vector pHIS2 and the full‐length cDNA sequences of OsMYB306 and OsRAV11 were inserted into the vector pGADT7. Co‐transformation of various combinations was performed according to the manufacturer's instructions. The selection of positive transformants was conducted on SD medium lacking Trp and Leu (DDO). The test of binding between the protein and different promoter fragments was conducted on SD medium lacking Trp, Leu and His (TDO) containing 50 mM 3‐amino‐1,2,4‐triazole (3‐AT). Primers used for vector construction are shown in Table S4.

4.3. Electrophoretic Mobility Shift Assay (EMSA)

The coding sequence of OsMYB306 was cloned into expression vector pET32a. The OsMYB306‐His recombinant protein was induced in Escherichia coli BL21 (DE3) using 0.2 mM IPTG (isopropyl β‐D‐1‐thiogalactopyranoside) at 15°C overnight and purified using Ni‐IDA Resin (Novagen). Oligonucleotides were synthesised and labelled with biotin. The unlabeled oligonucleotides were used as competitors. To perform the EMSA, a LightShift Chemiluminescent EMSA kit (Thermo Scientific) was used according to the manufacturer's instructions. Primers and probes used are listed in Table S4.

4.4. Dual‐Luciferase Reporter (DLR) Assay

The 2500 bp promoter sequence of OsT5H and its derived mutant versions were constructed into the pGreenII 0800‐LUC vector to serve as reporters. Full‐length cDNA sequences of OsMYB306 and OsRAV11 were cloned into the pGreenII 62‐SK vector to serve as effectors. To facilitate direct comparison with the GFP control and to assess transcriptional activation or repression under comparable conditions, equal amounts of effector constructs were used in all infiltrations. The effector and reporter pairs were co‐transformed into 3‐week‐old N. benthamiana leaves through Agrobacterium‐mediated infiltration (Hannah 1996). Activity of firefly luciferase (LUC) and renilla luciferase (REN) was assessed after 48 h using the Dual‐Luciferase Reporter Assay System Kit (Promega). The transcriptional activity was calculated as the ratio of LUC/REN. 35S::GFP was used as an internal control.

4.5. Gene Expression Analysis

For tissue‐specific expression analysis, total RNA was extracted from leaves, stems, panicles, florets and roots of wild‐type rice plants at the flowering stage, using the Plant RNA Kit (Omega). For developmental stage–dependent expression analysis, total RNA was extracted from leaves at the seedling, jointing and flowering stages of wild‐type rice plants.

For MD and SSB mock studies, rice shoot (at tillering stage) or stem (at flowering stage) segments (~2 cm, with the punched site in the middle) were collected for RNA extraction. For short‐term SSB challenge experiments, each rice tiller was infested with a single fourth‐instar SSB larva that had been starved for 2 h prior to infestation. Segments (~2 cm) with an apparent biting hole (where a larva penetrated into rice stem) were cut and used for RNA extraction. The time point when larval mouthparts began penetrating the rice stem (feeding onset) was defined as 0 h, and samples were subsequently collected at 1, 8 and 24 h post feeding onset.

Total RNA was reverse transcribed with Hifair III 1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) (Yeasen) following the manufacturer's instructions. RT‐qPCR was conducted with Hieff qPCR SYBR Green Master Mix (No Rox) (Yeasen) on a Light Cycler 480 real‐time system (Roche). The relative expression levels were calculated using the comparative 2^−ΔΔCt method (Livak and Schmittgen 2001) and normalised to the housekeeping gene OsActin. All primers used for gene expression analysis are listed in Table S4.

4.6. Subcellular Localisation

The full‐length CDS of OsMYB306 and OsRAV11 was amplified and cloned into pGreen‐GFP. For subcellular localisation in rice protoplasts, the recombinant plasmids were transferred into rice protoplasts using the PEG 4000 mediated rice protoplast transformation method (Bart et al. 2006). For subcellular localisation in N. benthamiana, the recombinant plasmids were first transformed into Agrobacterium tumefaciens and then injected into 3‐week‐old tobacco leaves. Fluorescence signals were observed 48 h post N. benthamiana infiltration or 16 h post protoplast transformation using a confocal laser scanning microscope (Olympus FV3000). DAPI (4′,6‐diamino‐2‐phenylindole) was added to visualise the nuclei.

4.7. Mechanical Damage and SSB Mock Treatment

For mechanical damage treatment, rice plants were punched in the shoot (at tillering stage) or stem (at flowering stage) using a hole puncher (2 mm in diameter), two punches per shoot/stem.

For SSB feeding mock analysis, SSB saliva was collected from 4‐ or 5‐instar SSB larvae. Selected larvae were first gently rinsed with sterile water, and then lightly stimulated their mouthparts using a micropipette tip to secrete saliva. The saliva was collected with a micropipette and stored at 4°C prior to use within 2 h. A total of 2.5 μL saliva was immediately applied to each punched site.

4.8. Quantification of Serotonin Levels in Rice

Serotonin levels in rice were quantified by HPLC as described previously (Kang et al. 2007) with some modifications. For mechanical damage treatments, rice samples were collected from the damaged region of tillers from 4‐week‐old plants at the indicated time point after treatment. For SSB‐related experiments, rice samples were collected from tillers of 2‐month‐old plants, and one ~2.5 cm segment surrounding the bitten site was excised from each infested tiller at the indicated time point after infestation. For the corresponding control groups without MD or SSB treatment, samples were collected from the corresponding positions on untreated tillers. Rice samples were homogenised in liquid nitrogen, and 100 mg tissue was transferred to 2 mL tubes and lysed in 1 mL 80% (v/v) methanol overnight. The homogenates were centrifuged at 14,000×g for 10 min at 4°C. The supernatants were filtered (0.45 μm filter) for HPLC analysis (Agilent 1290). Chromatographic separation was carried out on an Agilent Eclipse XDB‐C18 column (150 × 4.6 mm, 5 μm).

4.9. Immunoprecipitation‐Mass Spectrometry (IP‐MS) Assay

Total proteins were extracted from rice tissues, collected from plants including SSB infested plants (24 h post infestation), using Cell lysis buffer for Western and IP (Beyotime). Protein extracts were then incubated with OsMYB306‐His recombinant protein and Anti‐His magnetic beads (Biolinkedin) at 4°C for 4 h to facilitate protein binding according to the manufacturer's instructions; protein extracts incubated only with Anti‐His magnetic beads served as a blank control. Mass spectrometry (MS) analysis of the IP products was carried out by QLbio (Beijing, China).

4.10. Yeast Two‐Hybrid (Y2H) Assay

For Y2H assays, the coding sequences of OsRAV11 and OsMYB306 or its truncated forms were amplified and cloned into pGADT7 and pGBKT7, respectively. Y2H assays were carried out following the Yeastmaker Yeast Transformation System 2 (Clontech). Primers used for vector construction are listed in Table S4.

4.11. Bimolecular Fluorescence Complementation (BiFC) Assays

The full‐length coding sequences of OsMYB306 and OsRAV11 were cloned into pGreen‐based binary vectors to generate OsMYB306‐cEYFP and nEYFP‐OsRAV11 fusion constructs, respectively. The plasmids were transformed into Agrobacterium and then different combinations were co‐infiltrated into the epidermal cells of 3‐week‐old N. benthamiana leaves. The fluorescence signals were observed after 48 h incubation using a confocal laser scanning microscope (Olympus FV3000). DAPI (4′,6‐diamino‐2‐phenylindole) served as a nuclear marker.

4.12. Luciferase Complementation Imaging Assay (LCI)

Full‐length coding sequences of OsMYB306 and OsRAV11 were fused with the N‐terminus of luciferase or C‐termini of luciferase, respectively (OsMYB306‐nLUC and cLUC‐OsRAV11/OsRAV11‐nLUC and cLUC‐OsMYB306). The constructs were separately transformed into Agrobacterium. Agrobacterium with different combinations of vectors was co‐infiltrated into 3‐week‐old N. benthamiana leaves. After 48 h cultivation, signals were observed on a TANON Chemiluminescent Imaging system (Tanon 5200).

4.13. Co‐Immunoprecipitation (Co‐IP) Assay

For the Co‐IP assay, the constructs Ubi::OsMYB306‐4HA and Ubi::OsRAV11‐3Flag were generated and co‐transformed into rice protoplasts. After 16 h incubation in the dark, total proteins were extracted and subsequently incubated with anti‐FLAG magnetic beads (Sigma) at 4°C for 4 h. Both the total protein extracts (used as inputs) and the proteins immunoprecipitated by magnetic beads were detected by Western blot using anti‐HA (Sigma) and anti‐FLAG antibodies (Sigma).

4.14. Chromatin Immunoprecipitation Quantitative PCR (ChIP‐qPCR) Assay

ChIP–qPCR assay was performed as described (Wei et al. 2022) with modifications. Briefly, the construct Ubi::OsRAV11‐3Flag was transformed into rice protoplasts using the PEG 4000 mediated rice protoplast transformation method (Bart et al. 2006) and incubated for 16 h in the dark. Samples were fixed in 1% (v/v) formaldehyde for 10 min and quenched with 5 mM glycine. The nuclear extract was isolated and sonicated to obtain DNA fragments of 300–500 bp using a Covaris M220 (Covaris). Fragments were precleared with Dynabeads protein A/G (Invitrogen) for 1 h before incubation for 6 h with anti‐Flag magnetic beads (Sigma). The immunoprecipitated samples were used for qPCR analysis. The primers used for ChIP‐PCR are listed in Table S4.

4.15. SSB Bioassays

SSB artificial infestation was performed as described (Wang et al. 2023). In brief, rice tillers were detached from 2‐month‐old plants cultivated under standard paddy field conditions in Sanya. Tillers were placed into transparent glass test tubes (height 30 cm, diameter 3 cm), one tiller per tube with ~10 mL Yoshida rice nutrient solution.

For SSB performance assays assessing larval growth, survival and development, 10 newly hatched SSB larvae were transferred to one tiller. Ten days postinfestation (DPI), larval mortality was recorded and the individual weights of surviving larvae were measured; these were then individually transferred to a new tube containing a new, healthy rice tiller. This experiment was also performed at 15 DPI and 20 DPI with 10 biological replicates.

4.16. Statistical Analysis

Student's t‐test (p < 0.05) was used for significant difference analysis between two samples. One‐way analysis of variance (ANOVA) followed by Tukey's test (p < 0.05) was used for multiple comparisons. All the analyses were performed using GraphPad Prism (Version 8).

Author Contributions

Q.‐Y.S. planned and designed the research. J.‐R.Z., L.W. and Y.‐F.C. performed the research. X.‐H.G. contributed the bioinformatic analyses. Q.W. and Q.Q. generated the mutant lines. J.‐R.Z., M.J., Y.‐Y.T., A.M.R.G. and Q.‐Y.S. analysed the data and wrote the paper. All authors reviewed and approved the final manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No. 32072034) and in part by the Hainan Provincial Department of Science and Technology (ZDYF2025XDNY093).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Y1H analysis confirms a positive interaction between OsT5H promoter and TFs outlined in Table S2.

Figure S2: Subcellular localisation of OsMYB306.

Figure S3: Structural and sequence alignment of OsMYB306 and its mutant forms.

Figure S4: Relative expression of OsT5H in rice subjected to mechanical damage and SSB feeding.

Figure S5: FPKM values of transcription factors in response to SSB infestation.

Figure S6: Homology alignment of AtRAV1 and OsRAV11.

Figure S7: Subcellular localisation of OsRAV11.

Figure S8: Structural and sequence alignment of OsRAV11 and its mutant forms.

Figure S9: Temporal dynamics of OsT5H, OsMYB306 and OsRAV11 in response to mechanical damage.

Figure S10: Performance of SSB larvae fed on different materials.

Figure S11: Relative expression of OsT5H and 5‐HT content in the WT and mutant plants after SSB feeding.

Figure S12: Natural variation and haplotype analysis of OsT5H in the 3 k rice collection.

Figure S13: Growth phenotypes of WT and OsRAV11 overexpression lines.

Table S1: Genes revealed by sequencing of positive clones in a Y1H library screen using OsT5H promoter as bait.

Table S2: Transcription factors that could bind to the OsT5H promoter, as predicted by PlantRegMap.

Table S3: Potential transcription factors interacting with OsMYB306 identified by IP‐MS analysis.

Table S4: Primers used in this study.

Data Availability Statement

All the data generated or analysed during this study are presented in the manuscript.