OsCERK1 Interacts with OsHPP08 to Regulate Copper Uptake and Blast Resistance in Rice

doi:10.1016/j.rsci.2025.02.001

Abstract

Abstract:

The cell surface receptor chitin elicitor receptor kinase 1 (CERK1) is a well-known component of plant immunity. OsCERK1 is involved in regulating copper (Cu) uptake in rice, though the underlying mechanisms remain elusive. In this study, we identified proteins interacting with OsCERK1 and uncovered a novel heavy metal-associated domain-containing protein, OsHPP08. Our findings demonstrate that OsCERK1 phosphorylated and stabilized OsHPP08. Through structural analysis using AlphaFold, a yeast sensitivity assay of the Cu uptake-deficient yeast mutant, and Cu level measurements in oshpp08 mutants and overexpression plants (OsHPP08OE), we revealed that OsHPP08 facilitated Cu uptake. Additionally, rice seedling infection assays demonstrated that OsHPP08 positively contributed to blast resistance, with both OsCERK1 and OsHPP08 being essential for Cu-modulated blast resistance. Further analyses suggested that OsCERK1 and OsHPP08 likely enhanced blast resistance by regulating the antioxidant system and increasing H₂O₂ accumulation. In conclusion, OsCERK1 promoted Cu uptake by stabilizing OsHPP08, and together they contributed to Cu-modulated blast resistance, likely through the modulation of reactive oxygen species accumulation. These findings deepen our understanding of the intricate interplay between biotic and abiotic signals in rice.

Key words: Oryza sativa, copper, Magnaporthe oryzae, heavy metal ATPase domain, blast resistance, reactive oxygen species

Chen Ya, Liu Zhiquan, Yang Linyin, Wu Fujie, Cao Zijian, Shi Huanbin, Qiu Jiehua, Kou Yanjun. OsCERK1 Interacts with OsHPP08 to Regulate Copper Uptake and Blast Resistance in Rice[J]. Rice Science, 2025, 32(2): 203-216.

Figures/Tables 7

Fig. 1. OsHPP08 interacts with OsCERK1 both in vitro and in vivo. A, OsHPP08 structure with a single heavy metal-associated (HMA) domain is highlighted in yellow. B, Phylogenetic tree of OsHPPs in rice. Clustal W is used to compare the full-length protein sequences, and MEGA7.0 is used to construct the phylogenetic tree using the neighbor-joining method. C, OsHPP08 interacts with OsCERK1 in yeast. The positive and negative controls are PGADT7-T+PGBKT7-53 and PGADT7-T+PGBKT7-lam, respectively. SD (-WL) is a selective medium lacking leucine and tryptophan; SD (-WLHA) is a selective medium lacking leucine, tryptophan, adenine, and histidine; AD-E, The empty vector of PGADT7; BD-E, The empty vector of PGBKT7.D, OsHPP08 interacts with the intracellular domain of OsCERK1 (OsCERK1-IC) in the pull-down assay. GST/OsCERK1-His is used as the control, and OsHPP08-GST/OsCERK1-IC-His is used as the experimental group. GST, Glutathione S-transferase.E, Interaction of OsHPP08 with OsCERK1 in N.icotiana benthamiana using the firefly luciferase complementation assay. The indicated plasmid is transferred into Agrobacterium GV3101 and injected into N. benthamiana. After 48 h, the luminescence of the leaves is observed using a chemiluminescence imager. F, OsHPP08 interacts with the intracellular domain of OsCERK1 in the co-immunoprecipitation (Co-IP) assay. The Agrobacterium containing GFP/OsCERK1-Flag construct is co-injected into N. benthamiana as the control, and OsHPP08-GFP/OsCERK1-IC-Flag is co-injected as the experimental group. After 48 h, the protein of N. benthamiana leaves is extracted and incubated with GFP agarose beads at 4 ºC for 6 h to perform the Co-IP assay.

Fig. 2. OsCERK1 phosphorylates and stabilizes OsHPP08. A, OsHPP08 co-localizes with OsCERK1. The C-terminus of OsCERK1 is fused with mCherry, and the C-terminus of OsHPP08 is fused with GFP. OsCERK1-mCherry is transformed into rice protoplasts with OsHPP08-GFP. The Remorin-mCherry is used as a membrane marker, and the GHD7-CFP is served as a nucleus marker. Fluorescent observation reveals that OsHPP08 localizes to the membrane and the nucleus in rice, and co-localizes with OsCERK1 at the membrane. GFP, Green fluorescent protein; CFP, Cyan fluorescent protein. Scale bars, 10 μm. B, OsHPP08 is phosphorylated by OsCERK1. The recombinant plasmid OsHPP08-GST is co-expressed with OsCERK1-His and its empty vector pET-28a (His) in Escherichia coli BL21, respectively. Equal amounts of the GST purified recombinant proteins are detected by immunoblotting using indicated antibodies. p-OsHPP08-GST, Phosphorylated OsHPP08-GST; Phos-tag, Biotinylated Phos-tag zinc BTL111 complex. GST, Glutathione S-transferase.C, Degradation assay of OsHPP08-GST in the absence or presence of OsCERK1-His.

Fig. 3. OsHPP08 positively regulates copper (Cu) uptake in rice. A, Expression levels of OsHPP08 in different tissues of rice. The expression levels of OsHPP08 in the stem, root, leaf, and grain at the four-leaf stage are determined using qRT-PCR analysis. The Ubiquitin gene (LOC_Os03g13170) is served as an internal control.B, Expression levels of OsHPP08 under different Cu concentrations. TP309 is treated with nutrient solution containing 0, 0.2, 2, 20, and 50 μmol/L Cu ions for 12 h, respectively. The Ubiquitin gene is used as an internal control.C, Protein structure of OsHPP08 analyzed with AlphaFold. OsHPP08 contains a classic heavy metal-associated (HMA) domain structure, which may serve as a Cu metalloprotein. D, OsHPP08 has Cu uptake ability in yeast. Yeast monoclonal is diluted with ddH2O, further diluted 10, 102, 103, and 104 times, respectively. Subsequently, the diluted yeast is spot-coated on SD-Ura (galactose) medium, containing 0 or 0.5 mmol/L CuSO4 at 30 ºC for 3-7 d. E, Cu content in yeast. The yeast monoclonal of pYES2-Δcup2 and OsHPP08-pYES2-Δcup2 is cultured in SD-Ura medium (galactose) containing 0.05 mmol/L Cu ions for 2-3 d, and the Cu concentration in yeast is measured.F, Growth of the oshpp08 mutant and its wild type (TP309) before (-Cu) and after (+Cu) treatment with 5 mg/L Cu for 14 d. Scale bars, 1 cm.G, Cu concentration in the oshpp08 mutant and its wild type (TP309) with 5 mg/L Cu treatment. H-K, Fresh biomass (H), shoot length (I), root length (J), and root number (K) of the oshpp08 mutant and its wild-type before (-Cu) and after (+Cu) treatment with 5 mg/L Cu for 14 d.In A, B, E, and G, data are Mean ± SD (n = 3). Student’s t-test is used to analyze the data and generate P values. In H-K, data are Mean ± SD (n = 10). Different lowercase letters above columns indicate statistical differences at P < 0.05 by the Duncan test.

Fig. 4. OsHPP08 positively regulates rice basal resistance to Magnaporthe oryzae. A, Disease symptoms of OsHPP08 mutants and their wild type (TP309) following spray inoculation with M. oryzae. B, Relative fungal biomass in the representative leaves of oshpp08 mutants and TP309 after spray inoculation with M. oryzae. The relative fungal biomass is based on the DNA concentrations of M. oryzae Pot2 against the rice genomic Ubiquitin DNA level using quantitative PCR analysis.C and D, Expression levels of pathogenesis-related genes OsPAL1 (C) and OsNAC4 (D) after inoculation with M. oryzae. The Ubiquitin gene is served as an internal control.E, Disease symptoms of OsHPP08OE plants inoculated with M. oryzae. F, Relative fungal biomass of the representative leaves of OsHPP08OE plants. G and H, Expression levels of pathogenesis-related genes OsPAL1 (G) and OsNAC4 (H) of OsHPP08OE plants after inoculation with M. oryzae. I and J, Reactive oxygen species (ROS) accumulation dynamics in the leaves of the oshpp08 mutants (I) and OsHPP08OE (J) plants. Data are Mean ± SD (n = 3). Different lowercase letters above columns indicate statistical differences at P < 0.05 by the Duncan test.

Fig. 5. OsCERK1 and OsHPP08 contribute to Cu-induced resistance to Magnaporthe oryzae in rice. A and B, Disease symptoms of oscerk1 (A) and oshpp08 (B) mutants and their wild type Nipponbare (NIP) plants after spray inoculation with M. oryzae before (-Cu) and after (+Cu) treatment with 5 mg/L Cu for 14 d. C-H, Relative fungal biomass of the representative leaves of oscerk1 mutants (C), oshpp08 mutants (F), and their wild type plants after spray inoculation with M. oryzae before (-Cu) and after (+Cu) treatment with 5 mg/L Cu for 14 d. The expression levels of pathogenesis-related genes OsPAL1 and OsNAC4 in the oscerk1 (D and E) and oshpp08 (G and H) mutants after inoculation with M. oryzae. Data are Mean ± SD (n = 3). Student’s t-test is used to analyze the data and generate P values.

Fig. 6. Activities of antioxidant enzymes in Nipponbare (NIP), oshpp08, and oscerk1 mutants and their wild type seedlings before (-Cu) and after (+Cu) treatment with 5 mg/L copper (Cu) for 14 d. A and B, Content of hydrogen peroxide (H2O2) in oscerk1 and oshpp08 mutants treated with Cu. C and D, Activities of peroxidase (POD) in oscerk1 and oshpp08 mutants treated with Cu. E and F, Activities of catalase (CAT) in the oscerk1 and oshpp08 mutants treated with Cu. G and H, Activities of superoxide dismutase (SOD) in oscerk1 and oshpp08 mutants treated with Cu. Data are Mean ± SD (n = 3). Different lowercase letters above columns indicate statistical differences at P < 0.05 by the Duncan test.

Fig. 7. A proposed model illustrating that OsCERK1-OsHPP08 regulates copper (Cu) uptake to confer resistance against blast disease in rice. OsCERK1 likely phosphorylates and stabilizes OsHPP08 to positively regulate Cu uptake. The reactive oxygen species (ROS) accumulation triggered by Cu in the cell enhances the basal resistance against Magnaporthe oryzae in rice.

References 46

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