OsMAPK6 Phosphorylates and Degrades OsCATA to Regulate H₂O₂ Homeostasis Conferring Resistance to Bacterial Blight in Rice

ABSTRACT

Mitogen‐activated protein kinase (MAPK) cascades have been extensively reported to be involved in biotic and abiotic stress processes by regulating hydrogen peroxide (H₂O₂) homeostasis in plants. However, the detailed mechanisms by which MAPK cascades regulate H₂O₂ homeostasis in response to bacterial pathogens in rice remain largely unknown. Here, we demonstrate that rice CATALASE A (OsCATA) is a new substrate of OsMAPK6 and negatively regulates rice resistance to Xanthomonas oryzae pv. oryzae (Xoo) by directly suppressing H₂O₂ accumulation. Our results showed that OsMAPK6 interacts with OsCATA and specifically phosphorylates it at serine 460. The phosphorylation of OsCATA by OsMAPK6 attenuates OsCATA accumulation and reduces its catalase activity in vitro. The OsMAPK6‐overexpressing (OsMAPK6‐OE) plants showed lower catalase activity and higher accumulation of H₂O₂ compared with wild‐type plants. The oscata mutant plants generated by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR‐associated protein 9 (Cas9) strategy showed higher accumulation of H₂O₂ and increased resistance to Xoo. Together, these results suggest that the OsMAPK6‐OsCATA module is involved in modulating rice H₂O₂ homeostasis and immunity.

Rice OsMAPK6 phosphorylates and degrades OsCATA, an isoform of catalase, resulting in impaired OsCATA‐mediated H₂O₂ scavenging, which thereby triggers rice resistance to bacterial pathogens.

1.

Mitogen‐activated protein kinase (MAPK) cascades are widely involved in plant growth and development, biotic and abiotic stresses (Bi and Zhou 2017; Zhang et al. 2018). Plant MAPK cascades fulfil their physiological roles depending on the phosphorylation of downstream substrates (Bi and Zhou 2017). In rice, OsMAPK6 phosphorylates and activates two WRKY transcription factors, OsWRKY45 and OsWRKY53, to regulate rice resistance to fungal blast (Chujo et al. 2014; Kim et al. 2012; Ueno et al. 2015); OsMAPK4 phosphorylates two valine‐glutamine proteins, OsVQ14 and OsVQ32; and OsMAPK7 phosphorylates OsWRKY30 to regulate rice resistance to bacterial blight (Li et al. 2021; Jalmi and Sinha 2016).

H₂O₂ is one of the most important and stable reactive oxygen species (ROS). H₂O₂ at low concentrations can act as an important signalling molecule to regulate a wide range of physiological processes such as growth or development and biotic or abiotic stress responses. However, excess H₂O₂ can be toxic to plant cells and even cause cell death; thus, H₂O₂ content must be strictly regulated in vivo (Apel and Hirt 2004). Among H₂O₂ scavenging enzymes, catalase (CAT) is one of the most critical antioxidant enzymes, catalysing the decomposition of H₂O₂ into water and oxygen (Willekens et al. 1997). There are three catalase isoforms (OsCATA, OsCATB, OsCATC) in rice (Joo et al. 2014). Rice CATs have been functionally characterized to be involved in rice defence responses. Plants with loss of function of OsCATC accumulate excessive H₂O₂ with induced cell death, thereby triggering rice resistance to blast (Liao et al. 2022). Likewise, the oscatb mutant shows higher pathogenesis‐related (PR) gene expression and increased disease resistance (Gao et al. 2021). However, whether OsCATA has roles in plant immunity, and its potential regulatory mechanisms remain largely to be determined.

OsMAPK6 has been previously reported conferring rice resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). Although its upstream kinases have been identified (Chen et al. 2021; Wang et al. 2022), its downstream substrates are largely unknown. Activation of OsMAPK6 promotes H₂O₂ accumulation (Ma et al. 2021); however, OsMAPK6‐phosphorylated substrates that are responsible for H₂O₂ homeostasis remain to be identified, and the detailed mechanisms by which OsMAPK6 regulates H₂O₂ homeostasis in rice immune response remain to be determined.

OsMAPK6‐overexpressing (OsMAPK6‐OE) plants began to show reddish‐brown disease‐like lesions or spots on their leaves at the tillering stage (Figure 1a). When leaves were stained with 3,3′‐diaminobenzidine (DAB), which is used to indicate the accumulation of H₂O₂, the apparent red‐brown precipitates were observed in the OsMAPK6‐OE plants, but not in the wild type (Figure 1a). Consistently, the OsMAPK6‐OE plants had higher H₂O₂ contents than the wild type with or without Xoo infection (Figure 1b). In RNA‐seq assays, 3715 differentially expressed genes (DEGs), including 1973 up‐regulated DEGs and 1742 down‐regulated DEGs, were identified in the OsMAPK6‐OE plants relative to the wild type (Tables S1 and S2). The Gene Ontology (GO) term analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis together indicated that DEGs were mainly enriched in oxidation–reduction process and ROS metabolic process (Figure S1). Among the DEGs, 188 are involved in oxidation–reduction process and 22 encode peroxidase precursors (Table S3). Representative peroxidase precursors genes had altered expressions in the OsMAPK6‐OE plants compared to the wild type (Figure S2), supporting the concept that OsMAPK6 reprogrammes oxidation–reduction metabolism to promote H₂O₂ accumulation (All experimental methods are described in File S1).

FIGURE 1.

OsMAPK6 interacts and phosphorylates OsCATA. (a) 3,3′‐diaminobenzidine (DAB) staining for H₂O₂ accumulation in the leaves of OsMAPK6‐OE plants. Bars = 1 cm. (b) H₂O₂ contents in the leaves of OsMAPK6‐OE plants and wild type (WT). (c) Transcript levels of OsCATA and OsMAPK6 in rice leaves at the tillering stage after Xanthomonas oryzae pv. oryzae infection; water was used as the mock infection control. hpi, hours post‐infection. (d) Yeast two‐hybrid analysis of interaction between OsMAPK6 or OsMAPK3 and OsCATA. (e) Split‐luciferase assays showing the interaction of OsMAPK6 and OsCATA in Nicotiana benthamiana leaves. (f) Co‐immunoprecipitation assay to detect the interaction between OsMAPK6 and OsCATA in rice protoplasts. (g) OsMAPKK5‐activated OsMAPK6 phosphorylates OsCATA. Phosphorylation was detected by autoradiography. Values at the right side of the gel and blot indicate the molecular weight (kDa) of proteins. CBB, Coomassie brilliant blue staining. (h) Immunoblotting analysis of OsCATA levels in the leaves of WT and the OsMAPK6‐OE plants. Immunoblotting was performed with an anti‐OsCATA antibody and anti‐Actin antibody. Data represent means ± SD. n = 3 (b, c). Asterisks indicate a significant difference between the transgenic plants and WT determined by two‐tailed Student's t‐test at **p < 0.01. The different letters above each bar indicate statistically significant differences as determined by one‐way ANOVA followed by Tukey's multiple test (p < 0.05).

We performed immunoprecipitation mass spectrometry (IP‐MS) assays to mine the potential OsMAPK6 substrates that are responsible for H₂O₂ homeostasis. IP‐MS assays identified 25 unique proteins associated with OsMAPK6 by two biological replicates (Table S4). Of these, we were particularly interested in OsCATA because it is an isoform of catalase, one of the most critical antioxidant enzymes catalysing the decomposition of H₂O₂ (Willekens et al. 1997). OsCATA expression was activated upon Xoo infection, showing a similar expression pattern to OsMAPK6 (Figure 1c). The physical interaction between OsMAPK6 and OsCATA in vivo and in vitro was validated by yeast two‐hybrid assays (Figure 1d), split‐luciferase assays (Figure 1e) and co‐immunoprecipitation assays (Figure 1f). Moreover, OsMAPK3, a homologue of OsMAPK6 functioning in rice immunity (Kim et al. 2012), did not interact with OsCATA (Figure 1d,f). OsCATB and OsCATC, homologues of OsCATA, did not interact with OsMAPK6 (Figure S3). These data suggest the specific association between OsMAPK6 and OsCATA.

OsCATA contains six amino acid residues that could potentially be phosphorylated by OsMAPK6 (Figure S4). In vitro phosphorylation assays using prokaryotically expressed and purified recombinant proteins showed that non‐activated OsMAPK6, which lacks activity, could not phosphorylate OsCATA or OsCATC. However, OsMAPKK5‐activated OsMAPK6 could phosphorylate OsCATA, but not OsCATC (Figure 1g). This is consistent with the fact that OsMAPKK5 can phosphorylate and activate OsMAPK6 (Kishi‐Kaboshi et al. 2010). Using Phos‐tag gels followed by western blotting, the in planta phosphorylation assays further validated that OsMAPK6 could phosphorylate OsCATA because the phosphorylated OsCATA bands were observed clearly in OsMAPK6‐OE plants but not in the wild type (Figure 1h). Interestingly, the unphosphorylated OsCATA proteins were markedly weak in OsMAPK6‐OE plants relative to the wild type. Furthermore, total OsCATA levels were clearly lower in OsMAPK6‐OE plants than in the wild type (Figure 1h). In addition, both OsCATA levels and phosphorylated OsCATA showed the same pattern after Xoo infection (Figure S5). Taken together, these data suggest that OsMAPK6 may phosphorylate and degrade OsCATA.

We generated knockout mutants of OsCATA in rice cultivar Zonghua 11 (ZH11) by CRISPR/Cas9‐mediated mutagenesis and selected two homozygous mutants oscata‐1 and oscata‐2 containing large fragment deletions in the coding region (Figure S6). The oscata mutants that did not exhibit any off‐target events were backcrossed with the wild type to generate transgene‐free plants for subsequent analysis (Figure S6). The oscata‐1 and oscata‐2 mutants showed significantly increased resistance to all the tested Xoo strains, supported by shorter lesion length and less Xoo growth than the wild type (Figure 2a–c). To test if the increased resistance of oscata was attributable to the loss of OsCATA function, a genomic construct containing a 2‐kb native promoter and the coding sequence was transformed into homozygous oscata‐1 calli. Phenotypic analysis of the pOsCATA::OsCATA transgenic plants revealed a complete restoration of susceptibility to Xoo infection (Figure S6). Then, we crossed the oscata mutant with the osmapk6 mutant and isolated osmapk6 oscata homozygotes, finding that the osmapk6 oscata double mutant had comparable lesion length and Xoo population as the oscata mutant, exhibiting resistance to Xoo compared with the wild type (Figure 2d–f), while the osmapk6 mutant had longer lesion length and higher Xoo population than the wild type, suggesting that OsMAPK6 acts upstream of OsCATA and regulates rice resistance to Xoo by direct phosphorylation of OsCATA.

FIGURE 2.

OsCATA negatively regulates rice resistance to bacterial blight. (a) The responses of oscata mutants to different Xanthomonas oryzae pv. oryzae (Xoo) strains. Leaves of wild type (WT) and oscata mutants were inoculated with Xoo strains at the tillering stage. (b) Representative leaves of WT and oscata mutants after Xoo infection. (c) Growth of Xoo in leaves of WT and oscata mutants. cfu, colony‐forming unit. dpi, days post‐infection. (d) The responses of osmapk6, oscata and osmapk6 oscata mutants to two Xoo strains at the tillering stage. (e) Representative leaves of osmapk6, oscata and osmapk6 oscata mutants after Xoo infection. (f) Growth of Xoo in leaves of osmapk6, oscata and osmapk6 oscata mutants. (g) Catalase (CAT) activity in the leaves of the oscata mutants and OsMAPK6‐OE plants. (h) H₂O₂ content in the leaves of oscata mutants and WT 24 h after Xoo infection (hpi). Bars = 1 cm (b, e). Data represent means ± SD. n = 3 (c, f, g, h). n = 15 (a, d). Asterisks indicate a significant difference between the transgenic plants and WT determined by two‐tailed Student's t‐test at **p < 0.01 or *p < 0.05. The different letters above each bar indicate statistically significant differences as determined by one‐way ANOVA followed by Tukey's multiple test (p < 0.05).

Compared with OsCATB and OsCATC, the four unique residues of OsCATA, namely S92, T406, T409 and S460, may be phosphorylated by OsMAPK6. We therefore substituted each of these four residues individually for alanine and transformed constructs in which OsCATA was regulated by its native promoter into oscata‐derived protoplasts alongside an OsMAPK6‐overexpressing construct. The in vivo phosphorylation assay showed that the phosphorylated OsCATA only disappeared when S460 was substituted (Figure 3a), indicating that S460 may be the specific phosphorylation site for OsMAPK6. Then, we used glycosylase‐based T‐to‐G base editing to substitute OsCATA S460 (TCC) to alanine (GCC) in OsMAPK6‐OE plants (Kuang et al. 2025), generating an OsCATA ^S460A OsMAPK6‐OE plant (Figure S7). Similar levels of OsCATA expression were observed in both the OsMAPK6‐OE and the OsCATA ^S460A OsMAPK6‐OE plants. However, the total OsCATA level was clearly higher in the OsCATA ^S460A OsMAPK6‐OE plant than in the OsMAPK6‐OE plant. Accordingly, phosphorylated OsCATA was only observed in the OsMAPK6‐OE plant and not in the OsCATA ^S460A OsMAPK6‐OE plant (Figure 3b). Furthermore, we found OsMAPK6‐promoted phosphorylation of OsCATA was increased and OsCATA protein level was decreased in the OsMAPK6‐OE plant after Xoo infection, while phosphorylated OsCATA was not detected and OsCATA protein level was not changed in the OsCATA ^S460A OsMAPK6‐OE plant after Xoo infection (Figure 3c). Collectively, these data suggest that OsMAPK6 degrades OsCATA by phosphorylating it at S460. Furthermore, the lesion length of the OsCATA ^S460A OsMAPK6‐OE plant was longer than that of the OsMAPK6‐OE plant after Xoo infection (Figure 3d), suggesting that OsMAPK6‐mediated resistance largely depends on the degradation of OsCATA through its phosphorylation at S460. The prokaryotically expressed OsCATA has catalase activity (Chen et al. 2020). We here confirmed that the oscata mutants had lower catalase activity but higher H₂O₂ contents than the wild type (Figure 2g,h), confirming that OsCATA acts as a catalase in planta. The OsMAPK6‐OE plants exhibited decreased catalase activity and increased H₂O₂ content (Figure 2g), while the osmapk6 mutant had increased catalase activity and decreased H₂O₂ content, relative to the wild type (Figure 3e,f). However, the OsCATA ^S460A OsMAPK6‐OE plant had catalase activity and H₂O₂ content comparable to wild type, without disease‐like lesions (Figure 3g,h; Figure S7), further supporting OsMAPK6‐mediated degradation of OsCATA. In addition, flg22‐triggered production of ROS was significantly higher in the OsMAPK6‐OE plant or oscata mutant than in the wild type (Figure S8). Collectively, these results indicate that the phosphorylation and degradation of OsCATA by OsMAPK6 could impair the catalase activity in plants, resulting in the accumulation of H₂O₂.

FIGURE 3.

OsMAPK6 phosphorylates and degrades OsCATA. (a) Analysis of the putative phosphorylation residues of OsCATA by OsMAPK6. (b) Analysis of the specific phosphorylation residue of OsCATA by OsMAPK6. (c) Phosphorylation of OsCATA in the OsMAPK6‐OE and OsCATA ^S460A OsMAPK6‐OE plants after Xanthomonas oryzae pv. oryzae (Xoo) infection. (d) The responses of OsCATA ^S460A OsMAPK6‐OE and OsMAPK6‐OE plants to Xoo. Leaves of transgenic plants were inoculated with Xoo strains at the tillering stage. (e) Catalase (CAT) activity in the leaves of osmapk6 mutant. (f) H₂O₂ content in the leaves of osmapk6 mutant after Xoo infection. (g) Catalase activity in the leaves of OsCATA ^S460A OsMAPK6‐OE and OsMAPK6‐OE plants. (h) H₂O₂ content in the leaves of OsCATA ^S460A OsMAPK6‐OE and OsMAPK6‐OE plants after Xoo infection. Data represent means ± SD. n = 15 (d). n = 3 (e–h). Asterisks indicate a significant difference between the mutant and WT determined by two‐tailed Student's t‐test at *p < 0.05. The different letters above each bar indicate statistically significant differences as determined by one‐way ANOVA followed by Tukey's multiple test (p < 0.05).

Of the three catalases OsCATA, OsCATB, and OsCATC in rice, OsCATB and OsCATC are negatively involved in rice immunity (Gao et al. 2021; Liao et al. 2022; You et al. 2022), with their activity precisely modulated by their associating proteins. RESISTANCE OF RICE TO DISEASE1 (ROD1) modulates OsCATB to the plasma membrane, contributing to immune suppression (Gao et al. 2021). E3 ligase AvrPiz‐t Interacting Protein 6 (APIP6) interacts with and degrades OsCATC, promoting rice defence responses (You et al. 2022). Calcineurin B‐like interacting protein kinase 31 (CIPK31) associates with OsCATC and inhibits its activity, conferring rice immunity (Chen et al. 2023). Here, we demonstrated that OsMAPK6 phosphorylates and degrades OsCATA, resulting in impaired OsCATA‐mediated H₂O₂ scavenging, which thereby triggers rice resistance to Xoo. Compared with OsCATB and OsCATC, OsCATA has unique S/T residues that could potentially be phosphorylated by MAPKs, distinct subcellular localization (Gao et al. 2021), and a distinct expression pattern upon Xoo infection (You et al. 2022). Moreover, neither OsCATB nor OsCATC might compensate for the loss of function of OsCATA (Figure S9). These findings suggest that OsCATA, but not OsCATB or OsCATC, is the substrate for OsMAPK6 in response to Xoo infection. Although OsMAPK6 overexpression did not alter OsCATA expression, it did degrade OsCATA. The underlying mechanism of degradation should be investigated in future studies. In summary, OsMAPK6 either phosphorylates OsLIC to weaken the transcriptional inhibition of OsLIC toward OsWRKY30 (Wang et al. 2022), or phosphorylates and degrades OsCATA to reprogramme H₂O₂ homeostasis, suggesting the complex regulation for OsMAPK6‐mediated rice immunity.

Author Contributions

Jian Shi: methodology, formal analysis. Lihan Wang: methodology, investigation, data curation, software. Wenqian Zhu: methodology. Dan Chen: methodology, resources, visualization. Jie Chen: methodology, validation, data curation. Guixin Yuan: methodology, validation, project administration. Xingxun Liu: methodology. Yingshuang Liu: methodology. Meng Yuan: conceptualization, writing – original draft, writing – review and editing, funding acquisition, supervision.

Funding

This work was supported by grants from the National Natural Science Foundation of China (32572841) and the Fundamental Research Program of Hubei Province (2024AFE001).

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: GO and KEGG analysis of differentially expressed genes (DEGs). (a) GO enrichment analysis of Up‐DEGs. (b) GO enrichment analysis of Down‐DEGs. (c) KEGG analysis of Up‐DEGs. (d) KEGG analysis of Down‐DEGs.

Figure S2: Overexpression of OsMAPK6 promotes hydrogen peroxide accumulation. (a) Heatmap of differentially expressed genes involved in oxidation–reduction pathway. (b) The expression levels of OsPrx38, OsPrx113 and OsPrx114 in wild type (WT) and the OsMAPK6‐OE plants detected by reverse transcription‐quantitatve PCR assay. (c) The expression levels of OsCATA in WT and the OsMAPK6‐OE plants. Data represent means ± SD (n = 3). Asterisks indicate a significant difference between the OsMAPK6‐OE plants and WT determined by two‐tailed Student's t‐test at **p < 0.01. hpi, hours post‐infection.

Figure S3: Yeast two‐hybrid analysis of interaction between OsMAPK6 and OsCATB or OsCATC. BD‐53/AD‐RecT was the positive control. BD‐Lam/AD‐RecT was the negative control.

Figure S4: Analysis of His‐TF‐OsCATA and His‐TF‐OsCATC recombinant proteins. (a) Schematic diagram of protein structure of OsCATA, OsCATB, and OsCATC. S or T mean the potential phosphorylation sites. (b) Immunoblotting analysis of purified His‐TF‐OsCATA and His‐TF‐OsCATC recombinant proteins using anti‐His antibody. The fusion protein in the Ni Sepharose 6 Fast Flow was eluted with 250 mM imidazole three times, and E1, E2 and E3 indicate the first, second and third elution, respectively. Values at the right side of the gel indicate the molecular weight (kDa) of proteins. CBB, Coomassie brilliant blue staining.

Figure S5: Immunoblotting analysis of OsCATA levels in the leaves of the OsMAPK6‐OE plants after Xanthomonas oryzae pv. oryzae infection. Immunoblotting was performed with an anti‐OsCATA antibody and anti‐actin antibody. hpi, hours post‐infection.

Figure S6: Characterization of the oscata mutant. (a) Target sites designed for genome editing of OsCATA by CRISPR/Cas9. The target site nucleotides are shown in capital letters, and the PAM site is indicated as blue uppercase letters. (b) Gel electrophoresis of PCR products amplified over the targeted genomic region. (c) Sequence alignment of the target region between the oscata mutants and wild type (WT). (d) Sequence alignment of gRNA sites among OsCATA, OsCATB, and OsCATC. (e) Gel electrophoresis of PCR products of OsCATB and OsCATC amplified from the oscata mutants. (f) Putative off‐target sites detected in the oscata mutants. The putative off‐target sites in the whole rice genome were predicted using CRISPR‐P 2.0 (http://crispr.hzau.edu.cn/CRISPR2/). The red font represents mismatched bases and the blue font represents the PAM motif. (g) The responses of pOsCATA::OsCATA/oscata‐1 plants to Xanthomonas oryzae pv. oryzae strains. Leaves of WT and pOsCATA::OsCATA/oscata‐1 plants were inoculated with Xanthomonas oryzae pv. oryzae strains at the tillering stage. Bar = 1 cm.

Figure S7: Characterization of the OsCATA ^S460A OsMAPK6‐OE plant. (a) Sanger sequencing chromatograms of T‐to‐G base editing of OsCATA^S460A in the OsCATA ^S460A OsMAPK6‐OE plant. (b) Disease‐like lesions in the leaves of OsMAPK6‐OE plant. Bar = 1 cm.

Figure S8: Reactive oxygen species (ROS) accumulation after flg22 treatment. (a) ROS accumulation in leaves of OsMAPK6‐OE plant and wild type (WT) after flg22 treatment. (b) ROS accumulation in leaves of oscata mutant and WT after flg22 treatment. Rice leaf disks were treated with 10 mM flg22, with water as control. Data are means ± SD (n = 6).

Figure S9: Transcript levels of OsCATB and OsCATC in the oscata mutant leaves at the tillering stage after Xanthomonas oryzae pv. oryzae infection and water as a mock infection control. hpi, hours post‐infection.

Table S1: Detailed list of Up‐DEGs in OsMAPK6‐OE plants relative to wild type.

Table S2: Detailed list of Down‐DEGs in OsMAPK6‐OE plants relative to wild type.

Table S3: Detailed list of differentially expressed genes involved in oxidation–reduction process in OsMAPK6‐OE plants relative to wild type.

Table S4: The proteins identified in the OsMAPK6 complex by IP‐MS.

Table S5: Primers used in this study.

File S1: Materials and methods: plant materials and growth conditions, vector construction and rice transformation, off‐target detection, pathogen inoculation, RNA‐seq and data analysis, immunoprecipitation mass spectrometry, gene expression analysis, recombinant protein expression and purification, yeast two‐hybrid assays, split‐luciferase assays, co‐immunoprecipitation assays, in vitro phosphorylation assay, protein extraction and immunoblot analysis, physiological measurement, measurement of ROS, statistical analysis, primers used in this study.

Data Availability Statement

The RNA‐seq data from this study is deposited to NCBI Sequence Read Archive (BioProject ID: PRJNA843529).