The presence of calcium transport elements in the rice genome has been reported previously (Singh et al., 2014). To identify calcium signaling components involved in the rice immune response, we analyzed the expression patterns of different calcium transporter gene families, including cation/H⁺ exchangers (CAXs), autoinhibited Ca²⁺-ATPases (ACAs), annexins (ANNs), glutamate receptor-like channels (GLRs), and cyclic nucleotide-gated channels, in rice following M. oryzae infection and flg22 treatment. Upon infection with M. oryzae, the expression of genes belonging to the GLR and CNGC families was not induced, whereas several genes in the other three families were upregulated to varying degrees. Among them, the expression of OsCCX2, OsACA5, and OsANN3 was most strongly induced. Under flg22 treatment, several members of all five gene families were upregulated to varying extents, with OsCNGC16, OsCAX4, OsACA5, OsGLR1.1, and OsANN3 exhibiting the greatest degree of induction (Figures 1A, B). Taken together, these results indicate that several calcium-related genes respond to both M. oryzae infection and flg22 treatment. Although OsANN3 (LOC_Os05g31750) was also induced under both conditions, this gene has been previously characterized and shown to regulate ROS production and Ca²⁺ influx dynamics, and was therefore not further analyzed in this study (Zhang et al., 2021). Within the OsACA family, the expression of OsACA3, OsACA5, and OsACA7 was induced by both treatments. Among these genes, OsACA5 exhibited the highest expression level, whereas OsACA3 showed the second-highest expression. On the basis of their strong and consistent transcriptional responses, OsACA5 and OsACA3 were selected for further functional analysis.

To investigate the potential functions of OsACA3 and OsACA5, we performed a comprehensive analysis of the rice Ca²⁺-ATPase gene family, including an analysis of phylogenetic relationships and domain architecture, cis-acting element prediction, and spatiotemporal expression profiling (Figures 2A; Supplementary Figures S2, 3). While promoter cis-acting element analysis, spatiotemporal expression profiling, and conserved motif distribution analysis revealed considerable diversity among family members, domain architecture analysis revealed that OsACA3, OsACA5, and OsACA10 share an identical domain structure. This specific domain organization differs notably from that of OsACA6 and OsACA7; previous studies reported that OsACA6 is localized to the plasma membrane, whereas OsACA7 is localized to the Golgi apparatus (Huda et al., 2013; Singh et al., 2014). To further validate the subcellular localization of OsACA6, we co-expressed OsACA6–GFP with the ER marker mCherry–HDEL in rice protoplasts and performed a co-localization analysis (Supplementary Figure S5). OsACA6–GFP fluorescence was mainly detected at the cell periphery, whereas mCherry–HDEL showed a typical reticulate ER network pattern. Consistent with these distinct distribution patterns, only limited overlap between OsACA6–GFP and mCherry–HDEL signals was observed, supporting that OsACA6 is unlikely to be ER-localized. To verify the subcellular localization of the candidate genes, we constructed OsACA3–GFP and OsACA5–GFP fusion protein and transiently expressed them in rice protoplasts. The dataconfirmed that OsACA3 and OsACA5 are localized to the ER, which is consistent with the localization of the ER marker protein HDEL (Napier et al., 1992). Additional experiments revealed that the structurally similar gene OsACA10 is also localized to the ER (Figure 2B). Moreover, fluorescence intensity line-scan analysis along the indicated regions showed highly overlapping profiles between the GFP signal and the HDEL marker signal, with coincident peaks across the scanned distances (Figure 2C), further supporting ER localization of OsACA3, OsACA5, and OsACA10. Together, these results support the ER localization of OsACA3, OsACA5, and OsACA10. Given that OsACA10 shares an identical domain architecture with OsACA3 and OsACA5 (Figure 2A), we propose that this specific domain organization featuring the CaATP_NAI and ATPase_IIB_Ca domains may be associated with ER targeting in the OsACA family. Future studies are needed to test whether other members possessing this architecture (OsACA1, OsACA8, and OsACA9) are also ER-localized.

To explore the potential roles of OsACA3 and OsACA5 in rice immunity, we generated mutants on the NIP background, obtaining two independent mutant lines for each gene, namely, osaca5-1, osaca5-2, osaca3–1 and osaca3-2 (Supplementary Figure S1). Lesion areas were investigated seven days after wound inoculation with M. oryzae strain 70-15. The lesion area in NIP plants was significantly larger (by 40%) than that in osaca5 plants indicating that OsACA5 deficiency increases resistance to blast. Supsequent spray inoculation confirmed these results, with the diseased area in osaca5 plants reaching only nearly 15% of that in NIP plants (Figures 3A–D). Both the wound and spray inoculation experimental results revealed that osaca5 increases rice blast resistance.

In contrast, wound-inoculation assays of two independent osaca3 mutant lines (osaca3–1 and osaca3-2) revealed lesion areas that were not significantly different from those of NIP plants (Supplementary Figures S4A, B), indicating that OsACA3 deficiency does not alter rice blast resistance under these conditions. After establishing the resistant phenotype of osaca5, we assessed the transcript levels of other OsACA family members in osaca5.Compared with that of NIP, although the expression of most OsACA genes did not significantly change, and only minor decreases were observed for a few members, the expression of OsACA5 markedly decreased (Supplementary Figure S4C–K).

To clarify the role of OsACA5 in rice blast resistance, immune physiological indicators were detected in NIP and osaca5 plants after inoculation with 70-15. DAB and NBT staining at 48 hours post-inoculation revealed significantly larger areas stained dark brown and blue–purple in osaca5 plants than in NIP plants (Figures 3E–H). We also used chemiluminescence to assess the ROS burst in osaca5 plants after stimulation. Upon treatment with flg22, compared with NIP plants, osaca5–1 and osaca5–2 plants presented stronger ROS bursts (Figure 3I). We subsequently examined the expression levels of defense-related genes to further investigate the immune response and found that a series of defense-related genes were activated; the expression levels of Oryza sativa NAC domain-containing transcription factor 4 (OsNAC4), Oryza sativa WRKY transcription factor 45 (OsWRKY45), Oryza sativa pathogenesis-related protein 3 (OsPR3), Oryza sativa pathogenesis-related protein 1a (OsPR1a), and Oryza sativa phenylalanine ammonia-lyase 1 (OsPAL1) were significantly greater in osaca5 plants than in NIP plants at both 0 hours and 24 hours post-inoculation (Figures 3J–N). The increase in ROS activity, oxidative bursts, and defense-related genes expression in osaca5 plants upon pathogen inoculation indicate that OsACA5 negatively regulates the immune response in rice.

To investigate whether OsACA5 mediates Ca²⁺ influx under PAMP stimulation, noninvasive microtest technology (NMT) was used to detect dynamic changes in Ca²⁺ in mesophyll cells after treatment with PAMPs (flg22 and chitin), which have been shown to trigger PTI signaling in plants Chinchilla et al., 2007). Under untreated conditions, both NIP and osaca5 plants showed small Ca²⁺ fluxes, maintaining a stable state of Ca²⁺ influx/efflux. After the addition of 1 µM flg22, Ca²⁺ influx increased sharply. Compared with that in NIP plants, the Ca²⁺ flux in osaca5 plants was significantly greater, peaking rapidly within a short time and slowly returning to the initial state after 8 minutes (Figures 4A, B). After the addition of 1 µM chitin, Ca²⁺ influx increased sharply in both NIP and osaca5 plants, with the Ca²⁺ influx in osaca5 plants being significantly greater than that in NIP plants, although the intensity of Ca²⁺ influx was less than that after flg22 treatment (Figures 4C, D). Both flg22 and chitin caused Ca²⁺ influx, and the degree of influx was significantly greater in osaca5 plants than in NIP plants. Calcium influx is an early signal stimulated by PAMPs (Upadhyay, 2022). The stronger and more sustained Ca²⁺ influx observed in osaca5 plants may contribute to the heightened activation of immune responses and ultimately to the enhanced resistance of osaca5 plants to M. oryzae.

To validate the above results, we selected several calcium transporter genes for qRT–PCR analysis. We compared the expression levels of genes encoding calcium-dependent protein kinases (Oryza sativa calcium-dependent protein kinase 12 (OsCPK12) (Tavu and Redillas, 2025) and OsCPK20Fu et al., 2013)), calcium signaling sensors (Oryza sativa calmodulin-like protein 16 (OsCML16) (Liu et al., 2025) and Oryza sativa calcineurin B-like protein 2 (OsCBL2) (Qin et al., 2024)), and calcium channel proteins (OsCNGC9) (Wang et al., 2021), and calcium signaling-related genes (Oryza sativa phosphoinositide-specific phospholipase C1 (OsPLC1) (Li et al., 2017), Oryza sativa CBL-interacting protein kinase 31 (OsCIPK31) (Piao et al., 2010), and Oryza sativa receptor-like cytoplasmic kinase 185 (OsRLCK185) (Yamaguchi et al., 2013)) between NIP and osaca5 plants. Compared with those in NIP plants, the expression levels of OsCPK12, OsCML16, OsCIPK31, OsCBL2, and OsPLC1 significantly decreased in osaca5 plants, whereas the expression levels of OsRLCK185 and OsCNGC9 significantly increased. OsCPK20 expression did not significantly differ (Figure 4E), indicating that OsACA5 may indirectly influence the activity of other calcium channels.

An increase in disease resistance in plants is often accompanied by a reduction in yield (Kim et al., 2024b). Therefore, we investigated the agronomic traits of the osaca5 mutants. The height of the NIP plants was 84 cm, whereas those of the osaca5–1 and osaca5–2 plants were 76 cm and 78 cm, respectively. The height of the NIP plants was significantly greater than that of the osaca5 plants (Figures 5A, B). The seed setting rate of the osaca5 plants, below 60%, was significantly lower than that of the NIP plants (Figure 5C). The effective tiller number did not significantly differ between NIP and osaca5 plants (Supplementary Figure S6A). With respect to grain length, compared with NIP plants, osaca5 plants had significantly longer grains (Figures 5E, G). However, no significant difference in grain width was detected between the osaca5 and NIP plants (Figured 5F, S6B). Additionally, the thousand-grain weight of the osaca5 plants was significantly greater than that of the NIP plants (Figure 5D). A comparison of the total number of grains per panicle revealed that NIP plants produced far more grains than did osaca5 plants, with osaca5 plants did producing a greater proportion of empty grains (Figure 5H). These findings suggest that osaca5 mutation severely affects the seed setting rate in rice. However, no significant differences in pollen viability or other traits were detected (Supplementary Figures S6C, D).

To determine whether disrupted Ca²⁺ signaling contributes to the reduced seed setting rate of osaca5 plants, we examined the expression of several Ca²⁺ signaling- and fertility-related genes by qRT–PCR. These genes included the cyclic nucleotide-gated channel family members OsCNGC4 and OsCNGC13, as well as the Ca²⁺-dependent protein kinase family members OsCPK9, OsCPK21, and OsCDPK23. Previous studies have shown that OsCNGC4 and OsCNGC13 influence panicle fertility and the seed setting rate by regulating pollen germination and pollen tube growth within the pistil (Xu et al., 2017; Kim et al., 2024a), whereas OsCPK9, OsCPK21, and OsCDPK23 act as typical Ca²⁺ signal “decoders” that finely modulate reproductive processes such as pollen development, panicle development, and grain formation Asano et al., 2002; Wei et al., 2014; Wen et al., 2019). Compared with those in NIP plants, the transcript levels of OsCNGC4, OsCNGC13, OsCPK9, OsCPK21, and OsCDPK23 were significantly lower in both osaca5–1 and osaca5-2, with the two allelic mutants displaying a highly consistent downward trend (Figure 5I). These results indicate that Ca²⁺ signaling pathways that are closely associated with reproductive development are globally suppressed in the absence of OsACA5, which may be an important cause of the markedly reduced seed setting rate observed in the osaca5 mutants.

Transgenic lines were developed on the Nipponbare (NIP) background and maintained in our laboratory. Two independent osaca5 knockout mutants (osaca5–1 and osaca5-2) were generated using CRISPR/Cas9. Guide RNAs targeting OsACA5 were designed using the E-CRISP web tool and cloned and inserted into a Cas9 expression vector. Two sgRNAs were used to target OsACA5, and the resulting mutants contained indels at least two target sites, as shown in Supplementary Figure S1. The constructs were introduced into NIP calli via Agrobacterium tumefaciens EHA105-mediated transformation, and T₀ plants were screened by PCR and sequencing of the OsACA5 target site. T₁ and T₂ plants of the two knockout mutants and wild-type NIP were grown under field conditions at the Yazhou Bay Certification and Breeding Base in Sanya, Hainan Province. At maturity, the agronomic traits of ten randomly selected plants from each genotype were assessed. After harvest, the grains were dried at 37 °C for four days, after which the seed setting rate and thousand-grain weight were measured.

To investigate the evolutionary relationships and functional characteristics of the rice Ca²⁺-ATPase gene families OsACA and OsECA, we performed gene family identification and phylogenetic analysis, domain analysis, cis-acting element prediction, and spatiotemporal expression profiling for both families. First, a phylogenetic tree was constructed using the full-length amino acid sequences of members from the OsACA and OsECA families. The tree was generated in MEGA using the Neighbor-Joining (NJ) method, and its stability was evaluated by 1000 bootstrap resamplings. The resulting phylogeny reveals the evolutionary relationships among members of the two families and provides a basis for further functional inference. For domain analysis, the amino acid sequences of OsACA and OsECA proteins were analyzed using InterPro, SMART, and NCBI CD-Search (Conserved Domain Database, CDD) to identify conserved domains, such as the P-type ATPase domain and the CaATP_NAI domain. The domain information was integrated with the phylogenetic tree to further characterize the potential functional features of different gene members. In addition, cis-acting element prediction was conducted on the 2.0 kb promoter regions upstream of each OsACA and OsECA gene using the PlantCARE database. The identified cis-acting elements were categorized and summarized, and the results were visualized using bar graphs. Finally, to investigate the spatiotemporal expression patterns of the OsACA and OsECA family members, we retrieved expression data from the RiceXPro database. A heatmap was generated based on transcript abundance (TPM) across different tissues and developmental stages, revealing the dynamic expression profiles of these Ca²⁺-ATPase genes throughout rice development.

The coding sequence of OsACA5 lacking its stop codon was amplified and fused in frame to the N-terminus of GFP under the control of the CaMV 35S promoter in the pCAMBIA1390 vector. Recombinant and empty constructs were delivered into rice mesophyll protoplasts isolated from 10-day-old etiolated seedlings using a Plant Protoplast Preparation and Transformation Kit (Coolaber, Beijing, China; Cat. PPT111-5T) and transfected via a PEG-CaCl₂ solution at room temperature. After overnight incubation at 28°C in the dark, GFP fluorescence was observed in live protoplasts on a Zeiss LSM880 confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany).

Seven- to ten-day-old cultures of M. oryzae strain 70–15 were washed from agar plates with 0.5% (v/v) Tween-20, and the conidial suspension was adjusted to 2.0 × 10⁵ spores/mL. For the wound inoculation assays, 15 leaves per genotype from three-week-old rice seedlings were gently pricked with a sterile needle to generate three wound sites per leaf, and the wound sites were inoculated with 10 µL drops of the conidial suspension, after which the inoculation sites were covered with moist cotton soaked in a preservative solution. For the spray inoculation assays, two-week-old seedlings were uniformly misted with a conidial suspension (2.0 × 10⁵ spores/mL) prepared by eluting spores from culture plates with 5 g/L gelatin. In both assays, the plants were incubated in a moist chamber at 28°C in the dark for 24 h and then subjected to a 12 h light/12 h dark cycle at the same temperature and humidity for six days.

Total RNA was extracted from two-week-old Nipponbare seedlings spray-inoculated with M. oryzae strain 70-15 (treatment) or 5 g/L gelatin (mock control) after 24 h of incubation at 28°C in the dark (≥ 90% humidity) by grinding liquid nitrogen-frozen leaf tissue in TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and following the manufacturer’s protocol; RNA integrity was confirmed by 1% agarose gel electrophoresis, and the purity (A₂₆₀/A₂₈₀) was determined on a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). For resistance gene expression, osaca5 seedlings were processed similarly after 2 h of inoculation, and 1 µg of total RNA was reverse transcribed and preamplified in a single tube using HiScript IV All-in-One Ultra RT SuperMix (Vazyme, Nanjing, China). qRT–PCR was performed on a LightCycler 480 II (Roche, Basel, Switzerland) with ChamQ Universal SYBR qPCR Master Mix (Vazyme) in 20 µL reactions (1 µL of cDNA, 200 nM primers) using the following conditions: 95°C for 30 s and 40 cycles of 95°C for 10 s/60°C for 30 s, followed by melt-curve analysis; each of three biological replicates was assayed in technical triplicates.

Ca²⁺ movement in and out of rice mesophyll cells, known as Ca²⁺ flux, was measured in real time using noninvasive microtest technology equipment (NMT Physiolyzer, YoungerUSA LLC; Xuyue (Beijing) Sci. & Tech. Co., Ltd.). The leaf samples were torn, and the resulting pieces were fixed to the bottom of a culture dish with double-sided tape. Measuring solution was added to submerge the samples, and after a 30-minute incubation, the solution was discarded and replaced with 5 ml of fresh measuring solution for analysis. A mesophyll cell was identified under a microscope, and the Ca²⁺ flux microsensor was positioned approximately 10 μm from the cell surface before detection. Data were collected on each mesophyll cell for 5 minutes, and a treatment solution was added to the culture dish to reach a final concentration of 1 μM. Ca²⁺ flux was continuously monitored until the signal stabilized and did not significantly increase or decrease. Each test group consisted of 6 replicates. Ca²⁺ flux data were recorded using imFluxes V2.0 software (YoungerUSA LLC, Amherst, MA 01002, USA), and the expressed as picomoles • cm⁻² • s⁻¹; a positive value indicates efflux, and a negative value indicates influx.

To detect the accumulation of reactive oxygen species in rice following inoculation with M. oryzae, histochemical staining using DAB and NBT was performed to assess the localization of hydrogen peroxide (H₂O₂) and superoxide anion (O₂⁻), respectively, and quantify their concentrations. Rice leaves from two-week-old seedlings were harvested at 24 hours (for NBT) or 48 hours (for DAB) postinoculation with M. oryzae strain 70-15 (at a spore concentration of 1.5 × 10⁵/mL) and immersed in the respective staining solutions (DAB: 1 mg/mL, pH 3.5; NBT: 0.1%, pH 7.8). The samples were vacuum-infiltrated at –0.08 MPa to –0.1 MPa for 30 minutes and subsequently incubated in the dark at 25 °C for 8 hours (DAB) or 12 hours (NBT), after which the DAB-treated samples were gently agitated at 75 rpm. Chlorophyll was removed by boiling the leaves in 95% ethanol for 20 minutes. DAB-stained samples were imaged using a Smartzoom 5 stereomicroscope (Carl Zeiss AG, Oberkochen, Germany), where reddish-brown precipitates indicated sites of H₂O₂ accumulation, and the pixel intensity of the stained areas was quantified using ImageJ. NBT-stained samples were photographed with a smartphone, with blue deposits revealing O₂⁻ production, and the percentage of stained area per leaf was calculated in ImageJ using the formula: stained area (%) = (blue pixels area/total leaf pixels) × 100%.

ROS were detected using the luminol method. Rice plants grown on 1/2 MS medium for 10 days were selected, and sheaths approximately 3 mm in length were excised and equilibrated in ddH₂O for 12 hours. The sheaths were then treated with 1 µM flg22 in a solution containing 20 µM luminol (Sigma) and 10 µg/ml horseradish peroxidase (Sigma). The reaction was continuously monitored at 5-second intervals for 15 minutes using a microplate reader (Tecan, Switzerland).

Lesion area percentages and lesion lengths were measured on digital images using ImageJ software. Agronomic traits such as plant height and tiller number were recorded and compiled for statistical evaluation. Sample normality for agronomic trait measurements and lesion data was assessed by the Shapiro–Wilk test in GraphPad Prism 8.0. Quantitative datasets were subjected to one-way analysis of variance (ANOVA) to detect overall differences among treatment groups, followed by Dunnett’s multiple comparisons test to compare each treatment with the control and report multiplicity-adjusted p values. All the statistical analyses were performed using GraphPad Prism. Differences at p < 0.05 and p < 0.01 were considered significant and highly significant, respectively.