Light-induced OsLIKE1 phosphorylation enhances rice resistance against blast disease

Domain: biology
Published: 2025-12-23
Editorial outcome: major_revision

Abstract
Rice blast caused by Magnaporthe oryzae poses a major threat to world food supply. Conditions conducive to the disease include insufficient light. Our previous studies found that light-dependent phosphorylation of light-harvesting complex II protein (LHCB5) mediates rice immunity; however, the underlying mechanisms, in particular, the upstream regulators of LHCB5 phosphorylation, remain unknown. Here, we show that OsLIKE1 functions as a lectin receptor-like kinase (LecRLK) to play a critical role in rice resistance against M. oryzae. OsLIKE1 interacts with and phosphorylates LHCB5. In addition, we find two phosphorylation sites in the kinase domain of OsLIKE1 important for its auto-phosphorylation and phosphorylation of LHCB5. Moreover, we find that variation in light-induced OsLIKE1 expression is driven by specific single-nucleotide polymorphisms (SNPs) within the promoter region. These SNPs promote the auto-phosphorylation of OsLIKE1 to phosphorylate LHCB5 and activate its immune function against the blast fungus. Collectively, these findings elucidate the mechanism by which light regulates OsLIKE1-dependent defense in rice and support SNP-based rice breeding programs that increase disease resistance.

Introduction
Environmental conditions strongly influence pathogen–host interactions. Rice blast epidemics often occur under overcast and rainy conditions, not only due to the favorable environment for the pathogen but also because of insufficient light, which reduces host resistance1–3. Given the importance of breeding rice varieties resistant to blast disease, elucidating the mechanism of light-regulated rice immunity is a critical research goal. Previous studies have shown that light induces the phosphorylation of the light-harvesting complex II protein (LHCB5) upon infection by the rice blast fungus Magnaporthe oryzae. The phosphorylation of LHCB5 occurs in the cytoplasm, and the phosphorylation of the 24th threonine in its chloroplast transit peptide (cTP) can accelerate its transport from the cytoplasm to the chloroplast, thereby inducing the accumulation of reactive oxygen species (ROS) in the chloroplast and activating immunity2. Resistance governed by LHCB5 phosphorylation is linked to the progeny harboring the desirable haplotype promoter, establishing the genetic basis of LHCB5-regulated resistance mediated by phosphorylation2. However, the mechanism by which LHCB5 undergoes phosphorylation in response to light and pathogen infection remains unclear.

Receptor-like kinases (RLKs) play crucial roles in the growth, development, and immunity of plants. Plants deploy many RLKs as pattern recognition receptors that detect microbe- and host-derived molecular patterns, serving as the first layer of inducible immunity4,5. RLKs contain a variable ectodomain responsible for ligand binding, a single-pass transmembrane domain, an intracellular juxtamembrane domain, and a cytoplasmic kinase domain. The extracellular domains of RLKs are highly variable, allowing them to perceive a wide variety of signals4,6. Based on the structure of their ectodomains, RLKs are divided into different sub-groups, including lectin receptor-like kinases (LecRLKs), which can be further categorized into L-, G-, and C-types based on their extracellular lectin domains7,8. While many RLKs have been well characterized for their roles in PAMP recognition and plant defense, increasing evidence supports the crucial roles of LecRLKs in plant development and responses to abiotic and biotic stresses. For instance, LecRK-IX.2, a L-type LecRLK, acts as a positive regulator of PAMP-triggered immunity in Arabidopsis9. Similarly, LecRK-I.9 recognizes extracellular ATP signals and directly phosphorylates RBOHD, triggering immune response in Arabidopsis10. In rice, OsLecRK, a G-type LecRLK, was reported to confer plant defense against brown planthopper, blast disease, and leaf blight disease11. These studies demonstrated that LecRLKs contribute to plant defense against biotrophic pathogens.

Aside from biotic pathogen-induced stresses, plants often encounter various abiotic environmental challenges, such as salt, drought, heat, cold, and light. Several reports have demonstrated the involvement of LecRLKs in plant responses to salt stress, wounding, drought, and heat stresses12–16; however, little is known about how LecRLKs respond to light and their likely underlying molecular mechanisms. Here, we identified OsLIKE1 as a LecRLK that interacts with LHCB5 and found that OsLIKE1 undergoes auto-phosphorylation in response to light stimulation and pathogen infection, subsequently phosphorylating LHCB5 to enhance resistance to rice blast.

Results
OsLIKE1 interacts with LHCB5 on the plasma membrane
We have previously shown that light induces the phosphorylation of LHCB5 in rice upon M. oryzae infection2. To identify the likely kinases that phosphorylate LHCB5, we performed immunoprecipitation-mass spectrometry (IP-MS) using LHCB5 overexpressing rice plants (LHCB5-OE) inoculated with the wild-type M. oryzae strain Guy112. 121 RLK proteins containing the kinase domain were identified (Supplementary Data 1). We validated interactions between selected 40 RLKs and LHCB5 using the yeast two-hybrid (Y2H) assay. To minimize the effects of the extracellular and transmembrane domains on protein expression and interaction, we expressed only their kinase domains (KDs) in the yeast Saccharomyces cerevisiae. The Y2H assay showed that a LecRLK, Os03g60810, exhibits the most vital interaction with LHCB5 (Supplementary Fig. 1 and Fig. 1a). We named this protein OsLIKE1 (LHCB5 interacting kinase 1). OsLIKE1 encodes a protein of 701 amino acids with a signal peptide and a lectin domain at the N terminus, a KD at the C-terminal region, and a transmembrane domain (TM) situated between the lectin B and KD domains (Fig. 1b). To identify locations of the OsLIKE1-LHCB5 interaction, we first observed OsLIKE1 by transiently expressing the GFP-tagged OsLIKE1 in the rice protoplast. When stained with FM4-64 dye, green fluorescence colocalized with red fluorescence of FM4-64 (Fig. 1c). To substantiate this localization, we generated transgenic rice lines overexpression OsLIKE1 with a C-terminal GFP tag (OsLIKE1-OE) in the ZH11 background (Supplementary Fig. 2a), and isolated the plasma membrane, chloroplast, nuclear, and cytoplasmic proteins from OsLIKE1-OE rice plants. Immunoblot analysis using compartment-specific markers confirmed that OsLIKE1 is predominantly located in the plasma membrane fraction (Fig. 1d).

To further characterize the in vivo interaction between OsLIKE1 and LHCB5 during M. oryzae infection, we performed a time-course co-immunoprecipitation (co-IP) assay. Proteins precipitated with anti-GFP beads were detected with the anti-LHCB5 antibody only in OsLIKE1-GFP lines, while no interaction was observed in GFP-only controls. Notably, the OsLIKE1-LHCB5 interaction was dynamically regulated upon pathogen challenge, with the binding strength robustly increased at infectious stages (24 to 72 h) compared to non-infection (0 h). (Fig. 1e). Similar results were also obtained in Nicotiana benthamiana using either the KD domain-containing or full-length OsLIKE1 (Supplementary Fig. 3a and b). This in vivo interaction was further validated by the bimolecular fluorescence complementation (BiFC) assay in transient expression of rice protoplasts and N. benthamiana (Fig. 1e and Supplementary Fig. 3c). In this assay, OsLIKE1 tagged with N-terminal YFP (OsLIKE1-YFPN) and LHCB5 tagged with C-terminal YFP (LHCB5-YFPC) were co-expressed in host cells, and the results showed the occurrence of colocalization mainly at the plasma membrane (PM) (Fig. 1f).

OsLIKE1 phosphorylates LHCB5 to enhance resistance against rice blast
To examine whether LHCB5 is a substrate phosphorylated by OsLIKE1, we expressed and purified His-tagged OsLIKE1-KD and GST-tagged LHCB5 in E. coli. In vitro phosphorylation assays showed phosphorylation of LHCB5 by OsLIKE1-KD (Fig. 2a). Our previous study demonstrated that the LHCB5 T24 residue is the sole phosphorylation site, and that stimulation of this site (T24D) induced cell death in N. benthamiana2. Therefore, we transiently expressed full-length OsLIKE1, OsLIKE1-KD, LHCB5, LHCB5T24A and LHCB5T24D in N. benthamiana leaves. Cell death was observed in leaves co-expressed OsLIKE1 or OsLIKE1-KD with LHCB5 and LCHB5T24D, but not in those co-expressed OsLIKE1 or OsLIKE1-KD with LHCB5T24A (Supplementary Fig. 4). These results suggested that OsLIKE1 triggers the activation of LHCB5 phosphorylation and the immune response.

To further investigate whether OsLIKE1 contributes to blast resistance, we generated knock-out (OsLIKE1-KO) transgenic rice lines in the ZH11 background (Supplementary Fig. 2b). The OsLIKE1-KO lines #7 exhibited base deletion mutation, while lines #18 and #24 exhibited base insertion mutations (Supplementary Fig. 2b). In infection, OsLIKE1-OE rice lines showed strong resistance to M. oryzae, with significantly reduced lesion areas, whereas OsLIKE1-KO lines were more susceptible (Fig. 2b–d). No agronomically important traits differences were found among OsLIKE1-OE, OsLIKE1-KO, and the wild-type cultivar in the absence of M. oryzae infection (Supplementary Fig. 5). Moreover, compared to wild-type ZH11 plants, OsLIKE1-OE lines (#6, #18, and #20) maintained comparable plant height and yields upon M. oryzae infection. Strikingly, the OsLIKE1-KO lines (#7, #18, and #24) displayed more severe yield losses and stunted growth than infected ZH11, demonstrating that OsLIKE1 is essential for maintaining both productivity and plant architecture under blast stress (Supplementary Fig. 6).

Moreover, analyzed the phosphorylation of LHCB5 in ZH11, OsLIKE1-OE, and OsLIKE1-KO rice lines, both uninfected and infected by Guy11 using Mn2+-Phos-tag gel electrophoresis. We found that OsLIKE1 phosphorylation is induced by Guy11 infection in the OsLIKE1-OE compared to uninfected lines (Fig. 2e). Notably, LHCB5 phosphorylation was induced by Guy11 in the OsLIKE1-OE lines but not in ZH11 or OsLIKE1-KO lines (Fig. 2e). This result was also confirmed in the protoplasts of ZH11, OsLIKE1-OE, and OsLIKE1-KO rice lines treated with or without purified mycelial (PM) elicitors (Fig. 2f)2. Taken together, these findings demonstrated that OsLIKE1 enhances rice resistance to blast by phosphorylating LHCB5.

OsLIKE1 forms a homodimer that undergoes auto-phosphorylation to subsequently phosphorylate LHCB5
Receptor homodimerization or oligomerization is a common mechanism for ligand-mediated receptor activation and intracellular signal amplification17–19. Therefore, we tested OsLIKE1 self-interaction through GST pull-down and BiFC assays (Fig. 3a, b). Furthermore, we generated stable transgenic rice co-expressing OsLIKE1-GFP and OsLIKE1-Flag, with GFP and OsLIKE1-Flag co-expression serving as negative controls. Co-IP assays demonstrated that OsLIKE1-Flag was specifically detected in anti-GFP immunoprecipitates from OsLIKE1-GFP/OsLIKE1-Flag lines but not from control lines (Fig. 3c), confirming OsLIKE1 homodimerization in planta. Using native PAGE analysis, we found that KDs are mostly present in dimers (Fig. 3d). To examine which domains of OsLIKE1 might be involved in self-interaction, deletion mutants were made in the extracellular Lectin-binding (LectinB) domain and the intracellular KD. GST pull-down and Co-IP assays revealed that the deletion of KD, but not LectinB, nearly abolished self-interaction (Figs. 3e, f, and Supplementary Fig. 7).

To identify the amino acids involved in OsLIKE1 self-interaction, molecular modeling and docking analysis were carried out. The OsLIKE1 3D model was obtained from AlphaFold2 calculations, and most regions were confident with pLDDT > 90 (Supplementary Fig. 8a). ClusPro was used to predict that the interaction regions between KDs. Specifically, Loop 2 (L2), Loop 4 (L4), Loop 6 (L6), Loop 8 (L8), Loop 10 (L10), Loop 12 (L12), Loop 14 (L14), and β-strand 12 (β12) and the amino acid residues involved were predicted to form respective hydrogen bonds (Supplementary Fig. 8b–d). Moreover, GST pull-down and co-IP with alanine substitution were carried out to verify this predicted model. The results showed that mutations in L6 (positions 429, 430, and 432) and L14 (positions 566, 567, 571, and 573) nearly abolish the interaction and homodimer formation (Supplementary Fig. 9a and b).

To determine whether OsLIKE1 forms homodimer to facilitate auto-phosphorylation, we expressed and purified OsLIKE1-KD and performed an in vitro phosphorylation assay using Mn2+-Phos-tag SDS-PAGE. We observed a slower migrating band of OsLIKE1-KD in the presence of ATP compared to its absence, indicating that phosphorylation occurs in OsLIKE1-KD (Fig. 3h). Mutations in L6 and L14 nearly abolish this auto-phosphorylation (Supplementary Fig. 9c). To further identify the phosphorylation sites of OsLIKE1-KD, we performed mass spectrometry analysis with the above purified OsLIKE1-KD, which showed two differentially phosphorylated residues in T522 and T523 (Fig. 3g and Supplementary Fig. 10). Mutation of both residues to alanine (OsLIKE1-KDT522A/T523A), rather than a single mutation, prevented OsLIKE1-KD phosphorylation (Fig. 2h). Furthermore, mutated the phosphorylated residues in OsLIKE1-KD, and found that T523A and T523A/T523A mutations abolished the phosphorylation of LHCB5 by OsLIKE1-KD, but not T522A mutation (Fig. 3i). These results suggested that OsLIKE1 can form homodimers through its KD to facilitate auto-phosphorylation and LHCB5 phosphorylation.

The light-inducible promoter drives the expression of OsLIKE1 and promotes its auto-phosphorylation
Our previous studies have demonstrated that LHCB5 is subject to light-induced phosphorylation during infection of M. oryzae2. To further investigate whether the auto-phosphorylation of OsLIKE1 and its associated resistance were light-dependent, we examined OsLIKE1 phosphorylation in OsLIKE1-OE rice lines inoculated with Guy11 at different light intensities. Interestingly, we found that OsLIKE1-OE rice lines show enhanced resistance at various light intensities compared to ZH11 (Fig. 4a). Moreover, both OsLIKE1 and LHCB5 can undergo phosphorylation in OsLIKE1-OE rice lines infected by Guy11, and this phosphorylation showed no difference at 50 and 200 μmol photons m−2s−1 (Fig. 4b).

We speculated that this phenomenon may be related to the overexpression of OsLIKE1 and, further, different OsLIKE1 expression levels may exist in natural populations often associated with single-nucleotide polymorphisms (SNPs) within the promoter region. Therefore, we investigated the variance of OsLIKE1 promoter sequences from 3000 sequenced rice genomes20. SNPs were identified and separated into 20 groups using O. sativa ssp japonica cv. TP309 (SNP type 1) as the control, and most SNPs were distributed within types 1 and 2 (Supplementary Data 2 and Supplementary Fig. 11a). Notably, using Spearman’s rank correlation analysis to assess the relationship between disease resistance/susceptibility and SNPs across >400 rice accessions from the 3K dataset, we discovered a significant positive correlation between SNP type 2 and enhanced resistance (r=0.42, p<0.001; Fig. 4c). This finding further substantiates the evidence that OsLIKE1 promoter variations may play a key role in regulating immune responses.

To investigate the potential association between SNP type 2 and light-dependent OsLIKE1 transcription, we examined the transcription levels of OsLIKE1 in TP309 and YG456 rice varieties inoculated with or without M. oryzae at different light intensities. The results showed that a light intensity of 200 μmol photons m−2s−1, compared to 50 μmol photons m−2s−1, significantly induced OsLIKE1 expression in YG456. However, light intensity had no effect on OsLIKE1 transcription in the TP309 variety (Fig. 4d, e). Additionally, M. oryzae infection did not significantly induce the transcription of OsLIKE1 in either TP309 or YG456 at a light intensity of 200 μmol photons m−2s−1 (Fig. 4f). To determine the transcriptional dynamics of OsLIKE1 and LHCB5 in response to M. oryzae infection under varying light intensities, we analyzed their expression in rice cultivars ZH11 and TP309. We found a striking contrast: the transcription of OsLIKE1 remained largely unchanged across all conditions, indicating it is not induced by either pathogen infection or light (Supplementary Fig. 12a and b). In contrast, LHCB5 expression was significantly upregulated by both light exposure and pathogen challenge, with a particularly pronounced induction observed at 48 and 72 hpi (Supplementary Fig. 12c and d), a pattern consistent with previous reports2.

We further amplified the promoter sequences of OsLIKE1 from YG456 and TP309 and analyzed SNP types. The results showed that the SNPs of OsLIKE1 promoter in YG456 belong to type 2 (Supplementary Fig. 11a and Supplementary Fig. 13). To validate the correlation between OsLIKE1 promoter SNPs and expression levels, we expressed the GFP reporter gene driven by different promoters (p35S:GFP, pOsLIKE1type1:GFP and pOsLIKE1type2:GFP) in rice protoplasts at different light intensities. The result showed that constructs p35S:GFP and pOsLIKE1type1:GFP show no other levels of green fluorescence at 50 and 200 μmol photons m−2s−1, while pOsLIKE1type2:GFP shows a significantly higher level of green fluorescence at 200 μmol photons m−2s−1 than that at 50 μmol photons m−2s−1 (Supplementary Fig. 11b). Western blot analysis also showed a higher GFP protein level under the control of pOsLIKE1type2 promoter at 200 μmol photons m−2s−1 than at 50 μmol photons m−2s−1, indicating that type 2 SNPs in the OsLIKE1 promoter are indeed light-inducible (Supplementary Fig. 11c).

To validate the stability of these findings in rice, we generated stably inherited transgenic rice lines expressing OsLIKE1 gene driven by three distinct promoters: the ubiquitin promoter (pUbi), the SNP type 1 promoter (pSNP type 1), and the SNP type 2 promoter (pSNP type 2). The constructs pC1305:pUbi:OsLIKE1 and pC1305:pSNP type 1:OsLIKE1 exhibited comparable levels of green fluorescence at both 50 and 200 μmol photons m−2s−1. In contrast, pC1305:pSNP type 2:OsLIKE1 showed significantly higher fluorescence intensity at 200 μmol photons m−2s−1 compared to 50 μmol photons m−2s−1 (Fig. 4g). Identical results were observed when OsLIKE1 was replaced with the GFP reporter gene in transgenic rice lines (Supplementary Fig. 11d), confirming the light-dependent regulatory role of the SNP type 2 promoter.

We further selected 15 rice samples in 3k rice varieties to analyze SNP types and promoter transcription levels. We found that 12 of these samples belong to SNP type 2, and in these, the transcription and protein levels of OsLIKE1 were significantly upregulated under high light induction, compared to TP309 and SNP type 3 rice varieties (Fig. 4h and Supplementary Fig. 14a). All 12 rice samples notably showed OsLIKE1 auto-phosphorylation and LHCB5 phosphorylation (Fig. 4i and Supplementary Fig. 14b). Through genetic crosses between the resistant donor YG456 and susceptible LTH varieties, we previously demonstrated the stably inherited LHCB5 phosphorylation2. To validate whether the OsLIKE1 SNP type 2 promoter is heritable, we analyzed the OsLIKE1 promoter in progeny derived from LTH × YG456 crosses, which revealed that the previously reported blast-resistant progeny exhibiting LHCB5 phosphorylation exclusively carried the OsLIKE1 type 2 promoter variant (Supplementary Data 3). This finding demonstrated co-segregation of the OsLIKE1 type 2 promoter with heritable LHCB5 phosphorylation in the offspring. Together, these findings indicated that SNP-mediated promoter sequence divergence leads to different expression levels of OsLIKE1 in a light-dependent manner, which promotes OsLIKE1 auto-phosphorylation and LHCB5 phosphorylation.

Methods
Plant varieties
R4D1 rice varieties were obtained from Bo Zhou’s lab at the International Rice Research Institute. Yangeng 456 (YG456) was obtained from the Saline-Alkali Land Utilization and Research Institute, Liaoning, China. Oryza sativa japonica cv. ZH11 was transformed with constructs pCAM2300:OsLIKE1 and CRISPR-Cas9:OsLIKE1 with Agrobacterium tumefaciens-mediated transformation at EDGENE BIOT (Wuhan, China). The plants were grown in the greenhouse or fields at Nanjing Agricultural University in Nanjing, China.

Fungal strains and cultures
M. oryzae Guy11 was used as the wild-type strain in this study. The strains were cultured in a complete medium (CM). Liquid CM was employed to cultivate mycelia for DNA and RNA extraction. For conidiation, strain blocks were maintained on a straw decoction and corn (SDC) agar medium at 28°C for 7 days in the dark, followed by 3 days of continuous illumination under fluorescent light.

Resistance test and infection assays
For resistance tests, conidia were suspended to a concentration of 5×104 spores per milliliter in a 0.2% (w/v) gelatin solution. Five milliliters of this suspension were sprayed on two-week-old rice seedlings. The inoculated plants were then kept in a growth chamber at 25°C with 90% humidity, in the dark for the first 24 h, followed by a 16/8 h light/dark cycle. The disease severity was assessed at 7 days post-inoculation.

The disease severity of leaf blast under field conditions in the disease nursery was evaluated using the standard 0–9 scale45, with all assessments conducted in situ to reflect actual field infection patterns. The ten-level rating system was defined as follows: level 0 represented completely healthy leaves with no visible disease symptoms under field observation; level 1 showed initial infection signs with small brown spots less than 1 mm in diameter, typically appearing in field conditions; level 2 exhibited larger brown spots measuring 1–2 mm in diameter, as commonly seen in early field infections; level 3 presented characteristic circular to elliptical gray lesions 1–2 cm in diameter, representing typical field symptom development; level 4 demonstrated spindle-shaped lesions 1–2 cm in length confined between two veins, with infected area not exceeding 2% of the total leaf area under field assessment; level 5 displayed spindle-shaped lesions covering 2–10% of the leaf area, indicating moderate field infection; level 6 showed lesions affecting 11–25% of the leaf area, representing significant field damage; level 7 exhibited lesions occupying 26–50% of the leaf area, reflecting severe field infection; level 8 presented lesions covering 51–75% of the leaf area, indicating advanced field disease progression; and level 9 showed lesions affecting more than 75% of the total leaf area, representing maximum disease severity under field conditions.

RNA isolation and qRT-PCR
RNA isolation was performed using TRIzol (Invitrogen Life Technologies, Shanghai, China). cDNA synthesis was carried out with the PrimeScript™ RT Reagent Kit (TaKaRa). Quantitative RT-PCR was conducted using the ABI 7500 Fast Real-Time System, and transcript analysis was performed with the 7500 System SDS software. To compare the relative abundance of target gene transcripts in different rice varieties, the average threshold cycle (Ct) values were normalized to rice actin for each treated sample using the 2-ΔCt method, where ΔCt = (Ct_target gene - Ct_actin). Fold changes in gene expression among different rice varieties were calculated using the 2-ΔΔCt method, where ΔΔCt = (Ct_experiment - Ct_actin) - (Ct_control - Ct_actin).

Yeast two-hybrid assays
The OsLIKE1-KD cDNA was cloned into the pGBKT7 vector to create the bait construct. The prey construct was generated by cloning LHCB5 cDNA into the pGADT7 vector. Both constructs were confirmed by sequencing analysis and subsequently transformed into the yeast strain AH109 using the BD library construction & screening kit (Clontech). Tryptophan (Trp+) and leucine (Leu+) transformants were isolated and assayed for growth on SD-Trp-Leu-His (histidine)-Ade (adenine) medium.

In vivo co-IP assay
Co-IP using stable transgenic rice lines, rice protoplasts, and N. benthamiana cells was performed according to our previous study with minor modification2,46. Briefly, samples were extracted with IP buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.5% NP-40; 5 mM DTT; and protease inhibitor cocktail). The mixtures were incubated at 4°C with gentle shaking for 1 h. The IP complex was captured by adding 50 μl GFP-Trap® (ChromoTek, Germany, gta-20) or Anti-FLAG Antibody (Engibody, AT0022), followed by shaking at 4°C for an additional hour. The beads were recovered by centrifugation at 2500×g for 30 seconds and washed six times with cold TBS (50 mM Tris-HCl, pH 7.5; 150 mM NaCl). Then, 50 μl of glycine elution buffer (pH 2.5) was added to the beads. After boiling for 5 min, the samples were loaded onto SDS-PAGE gels for western blot analysis, and detected using anti-LHCB5 antibody (Agrisera, AS09407, 1:2000 dilution) and anti-GFP antibody (Abmart, M20004, 1:5000 dilution).

In vivo BiFC assay and subcellular localization
For subcellular localization in rice protoplasts, a cDNA fragment corresponding to the entire coding sequence of OsLIKE1 was cloned into the pCAM2300 vector to generate pACT1:OsLIKE1. OsLIKE1-YFPN and LHCB5-YFPC fusion constructs were generated and cloned into the pICH86988 vector; pICH86988-YFPN and pICH86988-YFPC vectors were used as controls. The fusion constructs were transformed into protoplasts prepared from TP309 seedlings, following a previously described method47. The recombinant plasmids were transferred to A. tumefaciens GV3101. The GV3101 strains carrying the indicated construct were mixed at OD = 0.4 each and infiltrated into leaves of 4-week-old N. benthamiana plants. Infiltrated leaves were observed 4 days later using a confocal laser scanning microscope.

In vitro pull-down assay
To construct GST- or His-fused plasmids, OsLIKE1-LectinB and OsLIKE1-KD were inserted into the pGEX4T-2 (GE Healthcare Life Science) or pET-32a (Navogen) vectors. The pull-down assay was carried out using the ProFound pull-down GST protein-protein interaction kit (Pierce) according to the manufacturer’s instructions45. Briefly, the GST-fusion protein was expressed in E. coli strain BL21 (DE3). Soluble proteins were incubated with 50 μl glutathione agarose beads (Invitrogen, 16101) for 1 h at 4°C. The beads were washed six times and then incubated with an equal amount of bacterial lysates containing His-fusion protein for another hour at 4°C. After washing the beads six more times, the presence of His-fusion proteins was detected by immunoblotting using an anti-His antibody (Abmart, M20001, 1:5000 dilution).

Production of polyclonal antibodies against OsLIKE1
To generate high-specificity polyclonal antibodies against OsLIKE1, we commissioned Abmart to synthesize and purify the custom peptide antigen (sequence: AVDQFLFPGFSGDGVATSGAAAVTSTGLLQLTNETKEVFGHGFYPKPVSLRNASTGAAASFSTTFVFAIVPKYPDAHGHGLAFALAPSVAVPGAVAGKYLGLFNTSDSTGQIKNKIVAVELDTARDDEFADIDDNHVGIDDNSLKSVNSSPAGYHDAATGGKLASVNLIGGEPLQVWIEYDGDSAWLEVTVSPAG) and perform rabbit immunization. Briefly, the immunization protocol consisted of 6 subcutaneous injections administered at 2-week intervals, using 200 μg of purified antigen per immunization emulsified with Freund’s adjuvant (complete adjuvant for primary immunization, incomplete for boosts). Serum was collected 7 days after the final boost and antibody titer was determined by ELISA, demonstrating strong immunoreactivity at dilutions exceeding 1:100. The antibodies specifically recognized an ~80 kDa band corresponding to OsLIKE1 in western blot analysis of rice protein extracts.

Phosphorylation assay
In vivo phosphorylation analysis was performed following a previously described method (2). Briefly, rice leaves were ground into powder in liquid nitrogen and resuspended in 1 ml of extraction buffer (10 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40), supplemented with 1 mM PMSF, 10 μl of protease inhibitor cocktail (Sigma, P8340), and 10 μl of phosphatase inhibitor cocktail 3 (Sigma, 524627). For in vitro phosphorylation analysis, His-OsLIKE1 and GST-LHCB5 were expressed in E. coli strain BL21 (DE3) (Sigma, CMC0014) and purified as previously described48. The reaction mixture was assembled by adding the following to microcentrifuge tubes: 0.5 μg of purified recombinant kinase OsLIKE1, 10 μg of potential substrate LHCB5, 3 μl of 10x reaction buffer (250 mM Tris, pH 7.5, 100 mM MgCl2, and 10 mM DTT), and water to a total volume of 27.5 μl. The reaction was initiated by adding 2.5 μl of 0.5 mM ATP (Sigma-Aldrich, A2383), mixed well, and incubated at 22°C for 1.5 h. To stop the reaction, 10 volumes of cold acetone were added.

Samples for phosphorylation analyses were resolved on 10% SDS-polyacrylamide gels prepared with 50 μM acrylamide-pendant Phos-tag ligand and 100 μM MnCl2, according to the instructions provided by the Phos-tag Consortium (Wako, AAL-107). The gels were electrophoresed at 80 V/gel for 3-6 h. Prior to transfer, gels were equilibrated in transfer buffer containing 5 mM EDTA for 20 min, twice, followed by equilibration in transfer buffer without EDTA for 10 min. Protein transfer from the Mn2+-Phos-tag acrylamide gel to the PVDF membrane was performed overnight at 80 V at 4°C, and the membrane was subsequently analyzed by Western blotting.

Subcellular fractionation
Isolate rice protoplasts from transgenic rice plant materials. For isolating protoplasts, a razor blade was used to cut the stems and leaves into 0.5 mm strips. They were then placed into a petri dish containing 10 mL enzyme solution (1.5% cellulase and 0.3% macerozyme). The vacuum was applied for 1 h for the infiltration of the enzyme solution. Plants were incubated for about 4 h in the dark with gentle shaking (40×g) at room temperature and the solution was removed with a glass pipet. 10 mL W5 medium was added and allowed gently swirl (80×g) for 1 h to release the protoplast. W5 Protoplasts were filtered through a 35 μm nylon mesh, transferred into an 8 mL glass vial, precipitated at 220×g for 4 min. These protoplasts will be used for subcellular fractionation. Membrane protein extraction: Using the Membrane and Cytosol Protein Extraction Kit (Beyotime, P0033), add Membrane Protein Extraction Reagent A to the rice protoplasts and homogenize on ice with 30-50 strokes to disrupt the cells. Centrifuge at 4°C and 14000×g for 30 min to collect the membrane fragments. Then add Membrane Protein Extraction Reagent B to fully extract the membrane proteins. Subsequently, centrifuge at 4°C and 14000×g for 5 min, and collect the supernatant as the membrane protein solution. The isolated membrane protein was used for western blot analysis with the H+ATPase antibody (Agrisera, AS07260). Nuclear and Cytoplasmic Protein Extraction: Rice protoplasts were resuspended in pre-cooled cell lysis buffer (10 mM pH 7.9 HEPES, 10 mM KCl, 1.5 mM MgCl2, 0.5% NP-40, 1 mM DTT), incubated on ice for 15 min, followed by the addition of NP-40 and another 10 min incubation on ice. The mixture was centrifuged at 300 g for 5 min to obtain the precipitate (nuclei), retaining both the supernatant and the precipitate. The precipitate was washed twice with nuclear wash buffer (10 mM pH 7.9 HEPES, 10 mM KCl, 1.5 mM MgCl2) and centrifuged at 800×g for 10 min to obtain purified nuclei. The isolated nuclear Protein was used for western blot analysis with the Histone H3 antibody (Abmart, P30266M). The supernatant was subjected to ultracentrifugation at 100000×g for 3 h, and the resulting supernatant was collected as the cytoplasmic protein fraction. The isolated cytoplasmic protein was used for western blot analysis with the Actin antibody (Abmart, T40104S). Chloroplast protein isolation: Rice leaves were soaked in 0.5% sarkosyl solution for 3-5 min, rinsed thoroughly, and ground using a mortar and pestle in 1.25 M NaCl, 40 mM HEPES (pH 7.6), 5 mM EDTA (pH 8), 0.1% bovine serum albumin, 0.1% β-mercaptoethanol following the high salt protocol. The homogenate was filtered through Miracloth and centrifuged in a microcentrifuge at 12000×g for 20 s. With large volumes, the homogenate was centrifuged at 3000×g for 5 min. The pellet was resuspended and washed twice in sorbitol dilution buffer (SDB: 0.33 M sorbitol, 20 mM HEPES, 2 mM EDTA, 1 mM MgCl2, 0.1% bovine serum albumin). The chloroplasts were loaded onto step gradients (30%: 70% Percoll in SDB) and centrifuged for 10 min at 4°C in a microcentrifuge. For large volumes, the step gradients were centrifuged for 30 min at 4°C at 1500×g. The chloroplasts were taken from the 30%: 70% Percoll interface, whereas the chloroplast excluded fraction remains on the top. Chloroplasts were shock-frozen in liquid nitrogen, resuspended in 50 μL of ice-cold protein extraction buffer, and centrifuged at 15000×g at 4°C for 10 min. The isolated chloroplast was used for Western blot analysis with the RbcL (Beijing Protein Innovation, AbP80037-A-SE) antibody.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The present manuscript of this group is the extension of their previous publication, wherein they described a SNP-mediated transcriptional increase in LHCB5 expression and its phosphortylation provide tolerance against rice blast by Magnaporthe. This manuscript described that OsLIKE1, a RLK is responsible for phosphorylation of LHCB5. Like LHCB5, a SNP-mediated transcriptional increase in OsLIKE1 promotes phosphorylation of LHCB5 to provide rice blast tolerance. The biochemical data in support of the hypothesis are very clean. However, neither in their previous paper, nor in this manuscript they showed a genome-wide association of these SNPs with the blast disease by scoring disease severity index using a scale of disease symptom and associating it with SNPs and providing r2 values. Biochemical data using a few lines and some transgenic or knock out lines are of course required for elucidating molecular mechanism, but, its application by the rice breeders requires a genome wide association of genomic variations with the pathogenesis and yield under pathogen pressure. I also like to know the performance of the OsLIKE1OE and OsLIKE1KO lines with respect to their yield and seed phenotypes under artificial infection by Magnaporthe. What is the genetic relationship between the SNPs of OsLIKE1 and OsLHCB5? Do they co-segregate with the blast tolerance? It appears from the figure 4G that 3-fold less transcriptional expression of OsLIKE1 totally abolishes phosphorylation of LHCB5. It is surprising and should be substantiated by the protein expression level and enzymatic activity of OsLIKE1 in those lines in comparison to the high OsLIKE1 expressing lines.", "reviewer_2": "This manuscript is a follow-up on a previous study from the same group (Liu et al., 2019) where they showed light-dependent immunity via LHCB5. The current manuscript studies the molecular interactions of the regulators identified in the previous study. The manuscript does not show if the exact mechanisms observed are indeed causal to the light effects on immunity.\n\nThere is very extensive knowledge on light-dependent signaling routes. I am puzzled by the lack of use of this information in this study. It would be straightforward to predict which LIKE1 promoter elements could be light-sensitive, and this would greatly help the functional rigor of the study.\n\nFollowing up from the previous point; if the aim is to understand how light regulates immunity via LHCB5 regulation, then there really is a need for investigation into how light is perceived to control LIKE1 expression. Since the light sensing research field is very well developed, this should not be too complicated (photoreceptors and their transcription factor targets are an obvious choice, chloroplast signals are an alternative, especially since LHCB5 itself will also localize to the chloroplast.\n\nOut of curiosity, verifying that it is really light itself that is relevant, or that adequate photosynthesis and thus provision of carbohydrates is key here.\n\nThe manuscript has an impressive dataset, but many of the interactions were studied in vitro or transiently in protoplasts. An example is the Like1-LHCB5 interaction in the first figures of the manuscript. It would be much better to include analyses in stable lines in the actual intact tissues where the interactions is supposed to be functional.\n\nUpon showing indications that LHCB5 and LIKE1 interact, the authors go on to study autophosphorylation of LIKE1. This does not flow naturally from the first part of the manuscript, and it is also not clear what question the authors intend to answer. It is not even clear why this section is in the manuscript at all.\n\nThe last part on SNPs in the LIKE1 promoter, lacks rigor. The authors have screened LIKE1 promoter sequences in a rice diversity panel, and transiently express different variants in protoplasts. There is no way with such a simplified system to study if these variations in expression have consequences for plant-level immunity. So again, the approach relies too much on transient expression systems.\n\nIt's a long stretch from the in vitro and transient expression studies, to the proposed model in Figure 5 and many more data from stable transgenic lines would be necessary to validate this.\n\nFigures show bar graphs. This is quite outdated and box plots, including their specific instructions on how to show individual data points, are the norm now.\n\nStatistical analyses need to be done better: t-test is not suitable for a design with two fixed factors, such as for example figure 4C-E. Better to use a 2-way ANOVA design.\n\nFigure 4F even has 16 individual t-tests done in one figure. I use Figure 4 as an example, but this problem is true for all figure panels with quantitative data"}, "author_response": "Point-by-point responses to referees\nReviewer #1\nThe present manuscript of this group is the extension of their previous publication,\nwherein they described a SNP-mediated transcriptional increase in LHCB5 expression\nand its phosphortylation provide tolerance against rice blast by Magnaporthe. This\nmanuscript described that OsLIKE1, a RLK is responsible for phosphorylation of\nLHCB5. Like LHCB5, a SNP-mediated transcriptional increase in OsLIKE1 promotes\nphosphorylation of LHCB5 to provide rice blast tolerance. The biochemical data in\nsupport of the hypothesis are very clean. However, neither in their previous paper, nor\nin this manuscript they showed a genome-wide association of these SNPs with the blast\ndisease by scoring disease severity index using a scale of disease symptom and\nassociating it with SNPs and providing r2 values. Biochemical data using a few lines\nand some transgenic or knock out lines are of course required for elucidating molecular\nmechanism, but, its application by the rice breeders requires a genome wide association\nof genomic variations with the pathogenesis and yield under pathogen pressure.\nResponse: We appreciate the reviewers’ constructive comments and suggestions. We\nacknowledge the importance of genome-wide association studies (GWAS) for linking\nSNPs to disease resistance in breeding applications. In the revision, we performed\nassociation analysis using field resistance data from over 400 rice accessions (3K\ndataset) and promoter variations of OsLIKE1. Our results revealed a significant\ncorrelation between SNP type 2 in the OsLIKE1 promoter and enhanced blast resistance\n(Spearman’s r = 0.42, p < 0.01; Figure 4C and Dataset S3). Notably, as highlighted in\nour response to Comment #3, genetic segregation analysis confirmed that the SNP type\n2 variant co-segregates with heritable LHCB5 phosphorylation and blast resistance in\nprogeny populations (Dataset S4), further validating its functional role in disease\nresistance inheritance. These findings are of particular importance for molecular\nbreeding, as it identifies a functional SNP marker (OsLIKE1 type 2 promoter) that can\nbe directly utilized to develop blast-resistant rice varieties. The stable inheritance of\nboth OsLIKE1 expression and LHCB5 phosphorylation further supports the robustness\nof this genetic mechanism in conferring durable resistance.\nI also like to know the performance of the OsLIKE1OE and OsLIKE1KO lines with\nrespect to their yield and seed phenotypes under artificial infection by Magnaporthe.\nResponse: We appreciate the reviewer’s constructive suggestions. We evaluated the\nagronomic performance of OsLIKE1-OE and OsLIKE1-KO lines under M. oryzae\ninfection. The OsLIKE1-OE lines maintained comparable plant height and yield to\nwild-type ZH11, while OsLIKE1-KO lines exhibited stunted growth and significant\nyield loss (Figure S6). These data underscore the role of OsLIKE1 in sustaining\nproductivity under disease stress.\nWhat is the genetic relationship between the SNPs of OsLIKE1 and OsLHCB5? Do they\nco-segregate with the blast tolerance?\nResponse: Through genetic crosses between the resistant donor YG456 and susceptible\nLTH varieties, we previously demonstrated the stably inherited LHCB5\nphosphorylation (1). We further analyzed the progenies from YG456 × LTH cross and\nfound that OsLIKE1 SNP type 2 co-segregated with LHCB5 phosphorylation and blast\nresistance (Dataset S4). This confirms a genetic linkage between OsLIKE1 promoter\nvariants and disease tolerance.\nIt appears from the figure 4G that 3-fold less transcriptional expression of OsLIKE1\ntotally abolishes phosphorylation of LHCB5. It is surprising and should be\nsubstantiated by the protein expression level and enzymatic activity of OsLIKE1 in\nthose lines in comparison to the high OsLIKE1 expressing lines.\nResponse: We thank the reviewer’s comments. To directly address the reviewer's\ninquiry regarding the threshold effect of OsLIKE1 expression on LHCB5\nphosphorylation, we performed comprehensive validation experiments using a custom-\ngenerated polyclonal antibody against OsLIKE1. Western blot analysis revealed a strict\ncorrelation between transcriptional levels, protein accumulation, and phosphorylation\nstatus: rice materials with low OsLIKE1 expression (SNP type 1/3) exhibited only weak\nOsLIKE1 protein signals and consequently no detectable LHCB5 phosphorylation. In\ncontrast, high-expressing lines (SNP type 2) consistently showed robust OsLIKE1\naccumulation and phosphorylation activity (Figure 4H and Figure S13). These results\nconfirm that OsLIKE1 expression must exceed a critical threshold to enable kinase\nfunction and downstream immune signaling.\nReviewer #2:\nThere is very extensive knowledge on light-dependent signaling routes. I am puzzled by\nthe lack of use of this information in this study. It would be straightforward to predict\nwhich LIKE1 promoter elements could be light-sensitive, and this would greatly help\nthe functional rigor of the study. Following up from the previous point; if the aim is to\nunderstand how light regulates immunity via LHCB5 regulation, then there really is a\nneed for investigation into how light is perceived to control LIKE1 expression. Since\nthe light sensing research field is very well developed, this should not be too\ncomplicated (photoreceptors and their transcription factor targets are an obvious\nchoice, chloroplast signals are an alternative, especially since LHCB5 itself will also\nlocalize to the chloroplast. Out of curiosity, verifying that it is really light itself that is\nrelevant, or that adequate photosynthesis and thus provision of carbohydrates is key\nhere.\nResponse: We greatly appreciate the reviewer's insightful suggestion regarding the\nlight-dependent regulation of OsLIKE1 expression. We fully agree that elucidating the\nlight perception pathway would significantly enhance the mechanistic understanding of\nour study. In response to this valuable suggestion, we conducted preliminary\nexperiments using the YG456 OsLIKE1 type 2 SNP promoter as bait in yeast one-\nhybrid screening against a rice nuclear cDNA library, identifying MYB2 as a\ntranscription factor that specifically binds to this promoter variant, with subsequent\nyeast one-hybrid assays confirming strong binding to the type 2 promoter (YG456) but\nnot the type 1 promoter (TP309) (see attached Fig. 1A). Importantly, as suggested, we\nexplored photoreceptor involvement through yeast two-hybrid assays, revealing that\nMYB2 interacts with phytochrome-interacting factor PIL12 (see attached Fig. 1B), a\nmember of a family known to interact with phytochromes (2-4), suggesting a potential\nlight signaling pathway: phyB-PIL12-MYB2→OsLIKE1 (type 2 promoter).\nHowever, as the reviewer would appreciate, fully characterizing this pathway requires\nextensive genetic evidence and molecular experiments. Given that the current\nmanuscript primarily focuses on identifying OsLIKE1 as the kinase responsible for\nlight-dependent LHCB5 phosphorylation and its role in blast resistance, we propose to\naddress the detailed light perception mechanism in a subsequent study. This approach\nwill allow us to maintain the clarity and focus of the current work while doing full\njustice to the complexity of the light signaling pathway.\nWe sincerely thank the reviewer for raising this important point, which has already\nguided our follow-up investigations and will undoubtedly strengthen our future work\non this system.\nFig. 1. (A) Yeast one-hybrid assay identifies MYB2 as a transcription factor specifically\nbinding to the OsLIKE1 type 2 promoter. The 1551bp OsLIKE1 promoter fragments\n(type 1 from TP309 and type 2 from YG456) were cloned into the pAbAi vector, while\nMYB2 was fused to the GAL4 activation domain (AD) in pGADT7. Positive\n(P53/RecT) and negative (empty pGADT7) controls are shown. Specific binding of\nMYB2-AD to the type 2 promoter (YG456) conferred aureobasidin A (AbA) resistance,\nwhile no growth was observed with the type 1 promoter (TP309) or empty AD vector.\n(B) Yeast two-hybrid assay demonstrating the interaction between PIL12 and MYB2.\nPIL12 was fused to the GAL4 activation domain (AD) in pGADT7, while MYB2 was\nfused to the GAL4 DNA-binding domain (BD) in pGBKT7. Positive (P53/RecT) and\nnegative (Lam/RecT) interaction controls are shown.\nThe manuscript has an impressive dataset, but many of the interactions were studied in\nvitro or transiently in protoplasts. An example is the Like1-LHCB5 interaction in the\nfirst figures of the manuscript. It would be much better to include analyses in stable\nlines in the actual intact tissues where the interactions is supposed to be functional.\nResponse: We sincerely appreciate the reviewer's suggestion regarding the need for in\nplanta validation of protein interactions. To address this important point, we have\nperformed comprehensive co-immunoprecipitation (Co-IP) experiments in stable\ntransgenic rice lines, providing robust evidence for both OsLIKE1-LHCB5 interaction\nand OsLIKE1 homodimerization. Using OsLIKE1-GFP transgenic rice and GFP-only\ncontrol plants, we found that anti-GFP immunoprecipitation specifically pulled down\nLHCB5 only in OsLIKE1-GFP lines, as detected by anti-LHCB5 antibody, while no\ninteraction was observed in GFP-only controls (Figure 1C). Furthermore, we generated\nstable transgenic rice co-expressing OsLIKE1-GFP and OsLIKE1-Flag, with GFP and\nOsLIKE1-Flag co-expression serving as negative controls. Co-IP assays demonstrated\nthat OsLIKE1-Flag was specifically detected in anti-GFP immunoprecipitates from\nOsLIKE1-GFP/OsLIKE1-Flag lines but not from control lines (Figure 3C), confirming\nOsLIKE1 homodimerization in planta. These results, now presented in the revised\nmanuscript, provide conclusive evidence that OsLIKE1 physically interacts with\nLHCB5 and forms functional homodimers at physiological expression levels in rice\ncells, significantly strengthening the physiological relevance of our findings.\nUpon showing indications that LHCB5 and LIKE1 interact, the authors go on to study\nautophosphorylation of LIKE1. This does not flow naturally from the first part of the\nmanuscript, and it is also not clear what question the authors intend to answer. It is not\neven clear why this section is in the manuscript at all.\nResponse: We appreciate the reviewer’s constructive feedback on the logical flow of\nour findings and have carefully restructured the manuscript to clarify the scientific\nrationale connecting the interaction between LHCB5 and OsLIKE1 to OsLIKE1’s\nautophosphorylation. In the revised version, we first establish the physical interaction\nbetween LHCB5 and OsLIKE1 (via co-IP, Y2H, and BiFC) and then demonstrate that\nLHCB5 is a phosphorylation substrate of OsLIKE1, showing that OsLIKE1 directly\nphosphorylates LHCB5 at the critical T24 residue, which is essential for immunity.\nOnly after confirming this kinase-substrate relationship do we investigate OsLIKE1’s\nautophosphorylation—a common regulatory mechanism for RLKs—as it represents a\nprerequisite for OsLIKE1’s kinase activity toward LHCB5 and a potential point of\nlight-dependent regulation. This sequential presentation (interaction → substrate\nphosphorylation → kinase activation) provides a clearer mechanistic progression,\nrevealing how OsLIKE1’s dimerization, autophosphorylation, and subsequent\nphosphorylation of LHCB5 collectively contribute to light-mediated immunity.\nThe last part on SNPs in the LIKE1 promoter, lacks rigor. The authors have screened\nLIKE1 promoter sequences in a rice diversity panel, and transiently express different\nvariants in protoplasts. There is no way with such a simplified system to study if these\nvariations in expression have consequences for plant-level immunity. So again, the\napproach relies too much on transient expression systems.\nResponse: We appreciate the reviewer's concern regarding the functional validation of\nSNP-dependent promoter activity and have addressed this by generating stable\ntransgenic rice lines expressing either OsLIKE1-GFP or GFP under the control of three\ndistinct promoters: the constitutive ubi promoter, the OsLIKE1 type 1 SNP promoter\n(from TP309), and the type 2 SNP promoter (from YG456). Western blot analysis of\nthese stable lines revealed that the type 2 promoter drove high OsLIKE1-GFP\nexpression under 200 μmol photons m⁻²s⁻¹ light but not under 50 μmol photons m⁻²s⁻¹,\nwhile the type 1 promoter produced only minimal protein expression under both light\nconditions (Figure 4G), mirroring results from parallel GFP-expressing lines (Figure\nS11D). This consistent pattern in stable transgenics confirms that the type 2 SNP variant\nconfers light-responsive expression at the protein level in planta, directly linking\npromoter variation to OsLIKE1 abundance and, consequently, its capacity to\nphosphorylate LHCB5 and enhance immunity—a relationship further supported by our\ncorrelation analysis between SNP type 2 in the OsLIKE1 promoter and enhanced blast\nresistance (Spearman’s r = 0.42, p < 0.01; Figure 4C and Dataset S3), and also the co-\nsegregation analysis of SNP type 2 with heritable blast resistance in progeny\npopulations (Dataset S4). These stable line experiments substantiate the biological\nrelevance of the promoter SNPs beyond transient assays, addressing the reviewer's valid\ncritique while advancing our understanding of how natural OsLIKE1 regulatory\nvariation modulates disease resistance.\nIt's a long stretch from the in vitro and transient expression studies, to the proposed\nmodel in Figure 5 and many more data from stable transgenic lines would be necessary\nto validate this.\nResponse: We fully acknowledge the importance of validating our proposed model\n(Figure 5) with stable transgenic evidence, and we have addressed this through multiple\nlines of rigorous experimentation. We generated stable transgenic lines expressing\nOsLIKE1-GFP or GFP under the control of the ubi promoter, type 1 SNP promoter, or\ntype 2 SNP promoter, demonstrating that only the type 2 variant confers light-\nresponsive protein expression (Figure 4G and Figure S11D). These findings were\nfurther supported by functional assays showing type 2 promoter-driven OsLIKE1 lines\nexhibit light-dependent blast resistance (Figure 4A-B) and genetic studies confirming\nco-segregation of SNP type 2 with heritable LHCB5 phosphorylation and disease\nresistance in progeny populations (Datasets S3-S4). Additionally, stable OsLIKE1-\nGFP/OsLIKE1-FLAG lines provided direct evidence for homodimerization in planta\n(Figure 3C). Together, these comprehensive stable transgenic analyses - spanning\nmolecular characterization (promoter activity, protein expression, and interaction) to\nphysiological validation (disease resistance and inheritance patterns) - provide robust\nmulti-level support for our model linking SNP variation to light-regulated immunity\nthrough the OsLIKE1-LHCB5 phosphorylation cascade.\nFigures show bar graphs. This is quite outdated and box plots, including their specific\ninstructions on how to show individual data points, are the norm now.\nResponse: We appreciated the reviewer’s comments. We have replaced bar graphs with\nbox plots showing individual data points and updated all statistical representations\naccording to current standards.\nStatistical analyses need to be done better: t-test is not suitable for a design with two\nfixed factors, such as for example figure 4C-E. Better to use a 2-way ANOVA design.\nFigure 4F even has 16 individual t-tests done in one figure. I use Figure 4 as an example,\nbut this problem is true for all figure panels with quantitative data\nResponse: We appreciate the reviewer's valuable guidance regarding statistical analysis.\nWe have thoroughly re-analyzed all quantitative data using appropriate statistical\nmethods, and the figure legends indicate the specific tests used."}, {"round": 2, "reviewer_comments": {"reviewer_1": "This study identifies OsLIKE1, a lectin receptor-like kinase, as a key player in rice defense by interacting with and phosphorylating LHCB5. Two critical phosphorylation sites in OsLIKE1 are essential for this activity. Light-induced expression of OsLIKE1 is driven by specific promoter SNPs, enhancing its immune function. However, I have the following questions, and author should explain in details.\n\n1. The author performed transient expression of OsLIKE1 and LHCB5 in rice protoplasts to quickly assess their subcellular localization and interaction. As now the stable OsLIKE1-GFP stable overexpressing line has been produced, a subcellular fractionation could be performed to reveal protein localization across cellular compartments (membrane, cytoplasm, nucleus, nucleolus).\n\n2. Is the interaction between OsLIKE1 and LHCB5 dependent on Magnaporthe oryzae infection? The authors demonstrated that LHCB5 phosphorylation increases upon M. oryzae infection, suggesting a pathogen-responsive regulatory mechanism. However, it remains unclear whether the physical interaction between OsLIKE1 and LHCB5 is also induced or strengthened under blast stress. Establishing whether their interaction is constitutive or specifically triggered by infection is important for understanding the dynamics of this defense signaling pathway.\n\n3. The author determined that LHCB5 T24 site is phosphorylated by OsLIKE1. Is T24 the only potential phosphorylation site in LHCB5? The author should show in vivo by IP and MS/MS (phosphoT24-specific antibody) that this is indeed the in vivo target site and there is no phosphorylation at this site in absence of OsLIKE1.\n\n4. What are the differences between the TP309, YG456, and ZH11 rice varieties? Why did the authors choose ZH11 specifically for generating stable overexpression, knockout (KO), and knockdown (KD) lines of OsLIKE1?\n\n5. What is the light intensity dependent expression profile of LHCB5 and LIKE1 in ZH11 and TP309 during M. oryzae inoculation.", "reviewer_2": "This manuscript has greatly improved since the previous submission and I applaud the authors for the major efforts put into this revised manuscript.\n\nAll points have bene resolved, except the one about how light signalling connects with the LIKE1 network. I believe that resolving this would make for a much better rounded paper. That being said, I also understand the author's point that they have already included such a lot of work and that they prefer to leave this to a putative separate story.\n\nWithout such data, the manuscript still stands as an interesting and valuable contribution on the molecular biology of LIKE-1-dependent immunity.\n\nThe authors might consider expanding their discussion on light-sensitive elements in the OsLIKE1 promoter to address the question if it is really light sensing itself, or the metabolic impact of the two different light intensities that controls the observed regulatory networks."}, "author_response": "Point-by-point responses to referees\nReviewer #1\nThe author performed transient expression of OsLIKE1 and LHCB5 in rice protoplasts\nto quickly assess their subcellular localization and interaction. As now the stable\nOsLIKE1-GFP stable overexpressing line has been produced, a subcellular\nfractionation could be performed to reveal protein localization across cellular\ncompartments (membrane, cytoplasm, nucleus, nucleolus).\nResponse: We thank the reviewer for this insightful suggestion. We have now\nperformed the recommended subcellular fractionation experiment using our OsLIKE1-\nGFP stable overexpression rice lines to precisely determine the protein's localization.\nCellular fractions—including plasma membrane, cytoplasm, chloroplast, and\nnucleus—were isolated and analyzed by immunoblotting with compartment-specific\nmarkers. The results clearly show that OsLIKE1-GFP is predominantly enriched in the\nplasma membrane fraction (Figure 1D, lines 103-112). This finding, derived from the\nstable transgenic material, is fully consistent with our initial transient expression assay\nin rice protoplasts, where OsLIKE1-GFP co-localized with the endomembrane marker\nFM4-64 (Figure 1C). The BiFC assay showed that the OsLIKE1–LHCB5 interaction\noccurs at the plasma membrane (Figure 1F). Together, these complementary lines of\nevidence strongly support the conclusion that OsLIKE1 is primarily a plasma\nmembrane-localized protein and that its interaction with LHCB5 takes place at this\nlocation. Thank you again for prompting this important clarification.\nIs the interaction between OsLIKE1 and LHCB5 dependent on Magnaporthe oryzae\ninfection? The authors demonstrated that LHCB5 phosphorylation increases upon M.\noryzae infection, suggesting a pathogen-responsive regulatory mechanism. However, it\nremains unclear whether the physical interaction between OsLIKE1 and LHCB5 is also\ninduced or strengthened under blast stress. Establishing whether their interaction is\nconstitutive or specifically triggered by infection is important for understanding the\ndynamics of this defense signaling pathway.\nResponse: We thank the reviewer for this insightful question regarding the dependency\nof the OsLIKE1-LHCB5 interaction on M. oryzae infection. We agree that\ndistinguishing between a constitutive and a pathogen-induced interaction is crucial for\nunderstanding the defense signaling dynamics. To address this question, we performed\na time-course co-IP assay using our OsLIKE1-GFP stable transgenic rice plants\nchallenged with M. oryzae at different time points. The results clearly demonstrate that\nthe physical interaction between OsLIKE1 and LHCB5 is significantly strengthened\nupon pathogen infection. While a basal level of interaction could be detected in mock-\ninoculated controls, the amount of LHCB5 co-precipitated with OsLIKE1-GFP\nmarkedly increased at specific time points post-inoculation (24 to 72 hours post-\ninoculation) (Figure 1E, lines 113-121). This finding provides direct biochemical\nevidence that the OsLIKE1-LHCB5 interaction is not merely constitutive but is\npositively regulated and enhanced during the plant's response to M. oryzae. This\npathogen-induced reinforcement of the interaction, coupled with the observed increase\nin LHCB5 phosphorylation, strongly supports a model where the OsLIKE1-LHCB5\ncomplex forms a key, dynamically regulated signaling module in rice blast resistance.\nThe author determined that LHCB5 T24 site is phosphorylated by OsLIKE1. Is T24 the\nonly potential phosphorylation site in LHCB5? The author should show in vivo by IP\nand MS/MS (phosphoT24-specific antibody) that this is indeed the in vivo target site\nand there is no phosphorylation at this site in absence of OsLIKE1.\nResponse: We thank the reviewer for raising this critical point regarding the specificity\nof the T24 phosphorylation site. Our previous work demonstrated that the\nphosphorylation of LHCB5 at threonine 24 (T24) is critical for initiating cell death (1).\nIn that study, we provided multiple lines of evidence demonstrating that T24 is the sole\nphosphorylation site of LHCB5, and that the T24A mutation completely abolishes\nLHCB5 phosphorylation in vivo. We have now clarified this central point in the revised\ntext to avoid any potential ambiguity (lines 139-141).\nIn the current study, we identified the upstream kinase responsible for this critical\nphosphorylation event, including the inability of OsLIKE1 to phosphorylate the\nLHCB5T24A mutant and the consequent loss of cell death induction (Figure S4), serve\nas independent genetic validation that OsLIKE1 functions precisely through this\nestablished, essential T24 site. We also showing that LHCB5 phosphorylation is\nundetectable in the OsLIKE1 knockout rice lines, establishing OsLIKE1 as the essential\nkinase for LHCB5 phosphorylation in planta (Figure 2). Collectively, evidence from\nboth our previous and current work consistently and unequivocally confirms that T24\nis the unique in vivo phosphorylation site of LHCB5, that its phosphorylation is\ndependent on OsLIKE1.\nWhat are the differences between the TP309, YG456, and ZH11 rice varieties? Why did\nthe authors choose ZH11 specifically for generating stable overexpression, knockout\n(KO), and knockdown (KD) lines of OsLIKE1?\nResponse: We thank the reviewer for these insightful questions regarding the selection\nof rice varieties and the specific use of ZH11 for genetic transformation.\nBoth ZH11 (Zhonghua 11) and TP309 (Taipei 309) are closely related blast-susceptible\njaponica rice varieties that share a high degree of genetic and sequence similarity. Given\ntheir comparable susceptibility and genetic background, the choice between them for\nstable transformation was primarily based on technical and practical considerations.\nCompared to TP309, ZH11 has been more extensively established and widely\nrecognized as a model system for genetic transformation in rice functional genomics,\nwith highly efficient, robust, and reproducible protocols (2, 3).\nIn contrast, YG456 is a blast-resistant japonica variety. There are two reasons for not\nselecting YG456 for generating stable overexpression lines. First, unlike ZH11, YG456\nlacks a well-established genetic transformation system, which poses a significant\ntechnical risk for the timely generation of transgenic materials. Second, and more\nimportantly, since YG456 already exhibits strong intrinsic resistance, overexpressing a\ndefense-related gene in this resistant background would be functionally redundant and\nbiologically uninformative, as it would not allow us to assess the gene's ability to\nenhance resistance in a high resistant background.\nTherefore, the use of the susceptible, transformation-optimized ZH11 background\nprovided a genetically uniform and functionally relevant system for definitively\ncharacterizing OsLIKE1’s role in blast resistance.\nWhat is the light intensity dependent expression profile of LHCB5 and LIKE1 in ZH11\nand TP309 during M. oryzae inoculation.\nResponse: We thank the reviewer for this question regarding the light-dependent\nexpression profiles of LHCB5 and OsLIKE1. We now systematically analyzed the\ntranscription levels of OsLIKE1 and LHCB5 in both ZH11 and TP309 rice varieties\nunder different light intensities and at various time points during M. oryzae inoculation.\nThe transcription of OsLIKE1 was stable and unresponsive to changes in light intensity\nor pathogen challenge in both ZH11 and TP309 (Figure S12, lines 247-254). We\nattribute this constitutive expression pattern directly to their promoter architecture. As\nestablished in our manuscript, both ZH11 and TP309 harbor the Type 1 SNP haplotype\nin the OsLIKE1 promoter, which we have demonstrated lacks light inducibility. The\ntranscription of LHCB5 was significantly induced by both light exposure and M. oryzae\ninfection. This inducible expression pattern is consistent across both ZH11 and TP309\nbackgrounds and aligns with our previous reports (1).\nReviewer #2:\nThis manuscript has greatly improved since the previous submission and I applaud the\nauthors for the major efforts put into this revised manuscript.\nAll points have bene resolved, except the one about how light signalling connects with\nthe LIKE1 network. I believe that resolving this would make for a much better rounded\npaper. That being said, I also understand the author's point that they have already\nincluded such a lot of work and that they prefer to leave this to a putative separate story.\nWithout such data, the manuscript still stands as an interesting and valuable\ncontribution on the molecular biology of LIKE-1-dependent immunity.\nThe authors might consider expanding their discussion on light-sensitive elements in\nthe OsLIKE1 promoter to address the question if it is really light sensing itself, or the\nmetabolic impact of the two different light intensities that controls the observed\nregulatory networks.\nResponse: We are deeply grateful to the reviewer for the positive and encouraging\nassessment of our revised manuscript, and for acknowledging the efforts we have\ninvested. We also sincerely thank the reviewer for this excellent suggestion to further\nrefine the discussion regarding the connection between light signaling and the\nOsLIKE1 network.\nWe have added a discussion of the potential mechanisms, including the role of the\nidentified light-sensitive SNP elements in the OsLIKE1 promoter and the possible\ninvolvement of light-mediated metabolic signals or photoreceptors in modulating this\npathway (lines 357-363)."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have addressed all the queries and concerns. The manuscript may be accepted for publication."}, "author_response": ""}]

Decision letter