Page 1/18 A Novel Non-Canonical Rectifying Calcium Channel in Rice Triggers Cell Death Mediated Robust Immunity Jun Wu  Hunan Agricultural University https://orcid.org/0000-0001-7351-6284 Jianbin Liu  Jilin Agricultural University Gui Xiao  Hunan Hybrid Rice Research Center Hai Liu  Huazhong Agricultural University Yi Liang  Hunan Agricultural University Zhaofeng Yi  Hunan Agricultural University Bai Bin  Hunan Hybrid Rice Research Center Xiushuo Liang  Huazhong Agricultural University Sheng Luo  Huazhong Agricultural University Jie Yang  IRRI-JAAS Joint Lab, and IRRI-CHINA Japonica Rice Research Center Shaowu Xue  College of Life Science and Technology, Huazhong Agricultural University https://orcid.org/0000- 0002-8838-3362 Wenxian Sun  Jilin Agricultural University https://orcid.org/0000-0001-6352-2461 Bo Zhou  International Rice Research Institute https://orcid.org/0000-0001-9631-954X Fang Yuan  https://doi.org/10.21203/rs.3.rs-6155589/v1 https://doi.org/10.21203/rs.3.rs-6155589/v1 https://orcid.org/0000-0001-7351-6284 https://orcid.org/0000-0002-8838-3362 https://orcid.org/0000-0002-8838-3362 https://orcid.org/0000-0001-6352-2461 https://orcid.org/0000-0001-9631-954X Page 2/18 Hunan Agricultural University Article Keywords: Posted Date: March 27th, 2025 DOI: https://doi.org/10.21203/rs.3.rs-6155589/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License.   Read Full License Additional Declarations: There is NO Competing Interest. https://doi.org/10.21203/rs.3.rs-6155589/v1 https://creativecommons.org/licenses/by/4.0/ Page 3/18 Abstract Plants utilize calcium as a signaling molecule to regulate innate immunity, including PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI), in addition to controlling growth and development1,2. Recent extensive research has highlighted that the activation of calcium ion (Ca2+) channels during PTI and the formation of Ca2+ channels during ETI are crucial for plant immunity3. However, comprehension on how crops substantially augment immunity through the enhancement of Ca2+ channel activity remains limited. Here, we report a rice lesion mimic mutant, called etd1 (elicitors triggered cell death 1), which also triggers cell death formation upon the challenge of rice blast elicitors. The recessive gain-of-function gene etd1 encodes a hypermorphic haplotype of OsCNGC13 which contains a single amino acid substitution of glycine-to-glutamate at the position of 483rd amino acid. The etd1 forms a novel non-canonical rectifying Ca2+ channel that significantly enhances Ca2+ influx. We position that etd1-driven excessive Ca2+ influx disrupts cellular calcium homeostasis, thereby triggering pathogen-induced cell death and conferring robust and broad-spectrum immunity to the rice blast pathogen. Introduction The etd1 mutant was previously identified in the screen of Pita2 suppressors derived from ethyl methanesulfonate (EMS) mutagenesis4–6. It exhibits typically development associated cell death phenotype (Fig. 1a-c; Supplementary Fig. 1a-j ). At the microscopic level, the etd1 mutant displays hallmark features reminiscent of hypersensitive response7, such as protoplast shrinkage, chromatin condensation, nuclear membrane blebbing, mitochondrial swelling, and chloroplast rupture (Fig. 1d; Supplementary Fig. 1k-m ). Given that spontaneous cell death mutants often enhance host resistance to pathogens8, we assessed the response of the etd1 mutant against rice blast. Unlike the typical compatible and incompatible reactions observed in IR64 (Fig. 1e, f), the etd1 mutant induces robust cell death without typical disease lesions covering over 80% of the leaf surface against both avirulent (V86010) and virulent (CA89) isolates (Fig. 1e, f). Interestingly, cell death was also induced by chitin or crude extracts of rice blast elicitors (Fig. 1g, h). The etd1 mutant despite exhibiting cell death phenotype, significantly enhances resistance to rice blast. To further investigate the resistance of etd1 against different M. oryzae isolates, we inoculated the etd1 mutant against 106 M. oryzae isolates collected from the Philippines and 14 major rice-producing regions in China (Supplementary Table. 1). The etd1 mutant exhibited evident cell death without disease lesions against all isolates whereas IR64 showed resistance to approximately 62.3% of isolates, demonstrating that etd1 confers robust broad-spectrum resistance against rice blast (Fig. 1i, j; Supplementary Table. 1). To investigate how etd1 confers resistance to rice blast, we inoculated leaf sheaths with CA89 and monitored its invasive progression in both IR64 and the etd1 mutant. At 12 hours post infection (hpi), approximately 75% of CA89 conidia formed appressoria and penetration pegs in IR64, compared to only 45% in the etd1 mutant (Fig. 1k, l). Invasive hyphae formation and subsequent Page 4/18 extension into adjacent cells over time were detected in IR64 but not in the etd1 mutant (Fig. 1k, l). Notably, in the etd1 mutant, abundant brown inclusions were detected around invasive hyphae starting at 24 hpi, followed by their continuous formation in neighbor cells, which are considered characteristics of a hypersensitive response9,10, whereas no such structures were observed in IR64 (Fig. 1k, l). Consistent phenomena were observed across tests with different isolates (Supplementary Fig. 2). Thus, at the microscopic level, cell death triggered in the etd1 mutant effectively suppresses pathogen invasion, thereby conferring broad-spectrum resistance to rice blast. Genetic analysis revealed that etd1 was controlled by a single genetic locus in a recessive manner (Supplementary Table. 2). Delimitation of etd1 via the bulked-segregant analysis by next-generation sequencing (BAS-seq) approach further identified a single genomic interval on chromosome 6 (Supplementary Fig. 3a), in which two non-synonymous mutations corresponding to two predicted gene models were identified (Supplementary Fig. 3b). Sequence verification of the PCR amplicons of these two gene models in the etd1 mutant and IR64 revealed that only the OsR498G0611825200.01 carries a true mutation in the etd1 mutant (Supplementary Fig. 3c, d). It corresponds to LOC_Os06g10580 in Nipponbare (NIP) encoding cyclic nucleotide-gated channel 13 (OsCNGC13), which is a known Ca2+- permeable inward channel11. Compared to LOC_Os06g10580 in IR64, etd1 contains a single nucleotide transition of G to A at the position of 1448 bp of the coding sequence, resulting in a glycine-to-glutamate substitution at the 483rd amino acid (Fig. 1m). Knockout of etd1 was sufficient to abolish the cell death observed in the etd1 mutant (Supplementary Fig. 4). On the contrary, the genetic complementation of etd1 in the OsCNGC13-nulled NIP established a typical cell death phenotype as appeared in the etd1 mutant (Fig. 1n-p; Supplementary Fig. 5). Thus, we conclude that etd1 is responsible for the spontaneous and triggered cell death in the mutant and represents a gain-of-function haplotype of OsCNGC13. The etd1 was found to localize to the plasma membrane (Supplementary Fig. 6), which is consistent with the subcellular localization of OsCNGC1311. The 483rd glycine (G483) residue of OsCNGC13 resides in the sixth transmembrane helix (S6) adjacent to the inner surface of the cell membrane (Supplementary Fig. 7c), which is a critical motif for Ca2+ channel gating12. Multiple sequence alignment of CNGC family in Arabidopsis and rice revealed that the G483 residue is complete conserved (Supplementary Fig. 7a-c), and the G483 residue in OsCNGC13 evolutionary clade across different plant species is also strictly conserved (Supplementary Fig. 7d, e), implying G483 is a critical residue for the Ca2+ channel mediated by CNGC13. Further, structural modeling revealed that the inter-residue distance between gating glutamine residues at position 486 in OsCNGC13 measures 10.65 Å and expands to 11.96 Å in etd1 (Fig. 2a). AlphaFold2 modeling also reiterated this finding, thus we hypothesize that the exchange of G483E possibly results in remarkable changes of channel activity (Supplementary Fig. 8). To investigate the Ca2+ channel activity conferred by etd1, we conducted a two-electrode voltage clamp assay. Following heterologous expressions in Xenopus laevis oocytes, OsCNGC13 and etd1 were also localized to the plasma membrane (Supplementary Fig. 9a). In contrast to the weak inward current observed for OsCNGC13 in a 30 mM Ca2+ bath, etd1 demonstrated a significantly enhanced Ca2+ transport capacity at 10 mM Ca2+ (Fig. 2b; Supplementary Fig. 9b-e). Moreover, the typical rectification property of OsCNCG13 Page 5/18 (z = 0.80 ± 0.07, V1/2=-138.98 ± 6.50) is evidently attenuated in etd1 (z = 0.72 ± 0.03, V1/2=-97.70 ± 3.28) (Fig. 2c). Treatment with the Ca2+-activated chloride-channel inhibitor—4,4 -disulfonic acid (DIDS13), results in a reduction rather than complete inhibition of the Ca2+-activated inward current in etd1 and OsCNGC13 (Supplementary Fig. 9f, g). We also tested the ion selectivity of the etd1 using monovalent cations K+ and Na+ and observed near background currents (Supplementary Fig. 9h, i). CNGCs typically function as channels in the form of homotetramers or heterotetramers14,15. Interestingly, co-expression of OsCNGC13 and etd1 in X. laevis oocytes significantly weakens the current changes caused by etd1, yet the rectifying attenuation induced by etd1 is not restored (Fig. 2b, c; Supplementary Fig. 9j, k). In summary, our findings demonstrate that the G483E mutation in etd1 alters the intrinsic rectification properties and channel activity of the original OsCNGC13, potentially by expanding the gating pore size. Additionally, we observed that the function of etd1 is impaired when co-expressed with OsCNGC13, shedding light on the possible mechanism underlying the recessiveness of etd1 in the background of OsCNGC13. The dynamics of Ca2+ influx in living plant tissues were then investigated through different assays. Non- invasive micro-test (NMT) analysis revealed that the etd1 mutant displayed significantly stronger Ca2+ influx than IR64 under the treatment of Ca2+ stimulation (Fig. 2d, e). Yellow cameleon calcium reporter analysis further illustrated that the etd1 mutant emitted much higher fluorescence resonance energy transfer signals than IR64 in live sheaths under the treatment of Ca2+ stimulation (Fig. 2f, g; Supplementary Fig. 11), indicating that the etd1 mutant is more responsive of cytosolic calcium ([Ca2+]cyt) accumulation upon the Ca2+ stimulation than IR64. Furthermore, stronger Ca2+ influx and elevated [Ca2+]cyt concentrations were observed in the etd1 mutant after the treatment of chitin and rice blast elicitors (Fig. 2h-k; Supplementary Figs. 10 and 11). The etd1 mutant also exhibited significantly stronger and prolonged [Ca2+]cyt accumulation at infection sites than IR64 at different time points post infection of M. oryzae (Fig. 2l). Thus, etd1 contributes to the robust Ca2+ influx in vivo. The findings of enhanced Ca2+ influx and cytosolic accumulation mediated by etd1 prompted us to further investigate the effect of external calcium ([Ca2+]ext) treatment on the induction of cell death in the etd1 mutant (Fig. 2m). The etd1 mutant started to initiate spontaneous cell death upon the exogenous treatment of 0.3 mM Ca2+ and above and a good correlation between the Ca2+ concentration and extent of cell death was further observed whereas no cell death was observed in IR64 under all treatments (Fig. 2n). The effect of [Ca2+]ext treatment on the response to the challenge of M. oryzae was also investigated. The induction of cell death observed in the etd1 mutant was also dependent on and correlated with Ca2+ concentration (Fig. 2o). Taken together, we concluded that calcium is required for etd1-dependent cell death in both processes of development and M. oryzae infection. The finding that etd1 mediated cell death is coupled with the impairment of agronomic traits raises a concern on the exploitation of etd1 in the rice breeding program. To further investigate the phenotypic plasticity of etd1, we undertook a near-saturated screening of etd1-suppressors (etds) from −diisothiocyana → stilbe ≠ − 2, 2 Page 6/18 approximately 500,000 M2 progeny derived from EMS mutagenesis. Three etd1 modifiers were identified, etds102, 145, 176, which correspond to amino acid substitutions G249R, L481F, E483K, respectively (Fig. 2p). All three etds mutants exhibited neither spontaneous nor triggered cell death phenotypes (Fig. 2q-t) while they recovered most agronomic traits compromised in the etd1 mutant. (Supplementary Table. 3). Further electrophysiological experiments revealed that these three etd1 modifiers exhibited similar Ca2+ channel activity as OsCNGC13 (Fig. 2u, v). It is worth noting that the etds176 substitutes glutamic acid in etd1 with lysine coincidently, reiterating the significance of E483 in terms of function (Fig. 2p). Nevertheless, identification of etds102 and 145 revealed that etd1 modifiers containing sequence mutations outside E483 can further regulate channel activity and affect the initiation of cell death as well as the restoration of agronomic traits. It is thus reasonable to speculate the existence of other etd1 modifiers which may decouple the cell death triggered by pathogen infection from the one developmentally regulated. In summary, we herein present a novel Ca2+ channel protein etd1 mediating broad-spectrum rice blast resistance by inducing cell death. etd1 exhibits non-canonical rectification inward Ca2+ channel activity, which is distinctive from known CNGC family proteins (Fig. 2b, c). Moreover, etd1 mediates a more robust Ca2+ influx, which was illustrated in living tissue assays upon the treatment of exogenous Ca2+, elicitors, and rice blast inoculation (Fig. 2d-l). The finding that mutations of residues outside E483 in etd1 modifiers undermine cell death and disease resistance to M. oryzae, indicating the existence of a regulatable structural basis involved in the function of etd1 (Fig. 2p-v). It is intriguing to investigate whether novel etd1 modifiers capable of retaining pathogen response while suppressing developmental associated cell death could exist through screening more etd1 suppressors. Moreover, in addition to etd1 modifiers, it is also expected to identify modifiers of the etd1 signalling components which can balance the robust response of etd1 to pathogen infection and development related cell death. Modification and engineering of etd1 and its signalling pathway may pave the way to explore the utilization of enhanced Ca2+ influx for breeding novel durable resistance mechanisms against rice blast. Interestingly, the cell death induced by etd1 resembles "calroptosis", a novel cell death mechanism triggered by hyperactivation of Ca2+ channels16–18. The etd1 mutant may serve as an ideal model germplasm for elucidating the mechanism of plant calroptosis. Materials and Methods Plant materials and growth conditions The etd1 mutant was initially identified from an EMS-mutagenized population of IR644, exhibiting both spontaneous and elicitors-triggered cell death phenotypes. The etd1 mutant and Nipponbare (NIP) were used for further transgenic analysis. To identify suppressors of etd1, a second round of EMS mutagenesis was performed on the etd1 mutant, generating approximately 25,000 M1 lines. Approximately 500,000 M2 progeny was then screened by treatment with crude extracts of rice blast elicitors to identify mutants that suppressed the elicitors-triggered cell death phenotype. To identify etd1 Page 7/18 modifiers, we crossed etds mutants with the OsCNGC13-nulled NIP and assessed F1 progeny for the presence or absence of the cell death phenotype. Sequencing the etd1 locus in parental lines that produced F1 progeny absence of cell death phenotype identified etds102, etds145 and etds176 carrying respective sequence mutations. All plants including transgenic rice plants were grown in the registered paddy fields in Changsha or in controlled growth chambers (14 h light at 30°C/10 h darkness at 28°C, relative humidity of ~ 80% and light intensity of ~ 1000 µmol m− 2 s− 1) for phenotypic analysis or pathogen inoculation. To assess the impact of [Ca2+]ext application on cell death phenotype of the etd1 mutant, germinated seeds were hydroponically grown in Yoshida’s cultural solutions19 with varying Ca2+ concentrations (0.01, 0.1, 0.3 [standard], 0.5, and 1 mM). After three weeks of cultivation in the growth chamber, phenotypic analysis and pathogen inoculation were performed, with the nutrient solution replaced every three days throughout the growth period. M. oryzae growth, elicitors extraction and infection assays In addition to CA89, V86010, S5 and 4029-1, all M. oryzae field isolates utilized in this study are detailed in Supplementary Table 1. All isolates are stored at -20°C on desiccated filter papers and grown under a 12 h photoperiod at 25°C for 10 days on prune agar medium (three pieces L− 1 prunes, 5 g L− 1 alpha- lactose monohydrate, 1 g L− 1 yeast extract, 1.5% agar) to produce spores for infection assays. The IR64- compatible isolate CA89 was used for extracting the crude elicitors. Briefly, 2 g of agar pieces containing CA89 mycelia were added to 1 L of Complete Medium (CM) and cultured at 28°C, 220 rpm for 3 days. The culture was filtered through a 0.45µm nylon membrane, autoclaved, and centrifuged to obtain the supernatant as the rice blast crude elicitors. Rice blast spray inoculation assays, leaf sheath injection inoculation assays, and elicitors assays were performed as described previously20. For spray inoculation, conidia were suspended 0.5% (v/v) Tween 20 at 5 × 10⁴ spores mL− 1, and 5 mL was sprayed onto 3-week-old rice seedlings using a compressed air sprayer. For sheath injections inoculation, conidia were adjusted to 1 × 10⁵ spores mL− 1 and injected into rice stems using a 1-mL syringe. For elicitors assays, chitin (N-acetylated chitoheptaose, SCN07, Qingdao HEHAI Biotech Co., Ltd) and rice blast elicitors with 0.5% (v/v) Tween 20 were sprayed onto seedlings. Treated plants were incubated in darkness (100% RH, 25°C) for 24 hours and subsequently transferred to a greenhouse (70–90% RH, 25–28°C). Five days of post-inoculation, the youngest fully expanded leaves at the time of inoculation were collected and scanned for symptom analysis. To assess lesion or cell death progression, ImageJ software (https://imagej.net/) was used to calculate the percentage of lesion or cell death area relative to the total leaf area, based on color contrast between healthy and diseased tissue. BSA-seq based cloning Page 8/18 To map the etd1 gene, we generated an F2 population of approximately 400 individuals by crossing etd1 with CO39. Equal amounts of leaf tissue from 50 randomly selected F2 individuals displaying spontaneous cell death (cell death pool) and 50 healthy F2 individuals (non-cell death pool) were pooled, and DNA was extracted to construct two DNA pools for bulked-segregant analysis by next-generation sequencing (BSA-seq). After sequencing, clean reads were aligned to the indica rice R498 reference genome (https://ricerc.sicau.edu.cn). The SNP-index, representing the ratio of mutant base reads to total reads, was calculated for both pools. To map candidate regions linked to spontaneous cell death, we derived the ΔSNP-index by subtracting the non-cell death pool SNP-index from the cell death pool SNP- index, with higher ΔSNP-index values indicating stronger genetic associations with the phenotype. Plasmid construction and plant transformation To generate the CRISPR/Cas9-based knockout construct, an 18 bp gene-specific sequence targeting the first exon of OsCNGC13 was synthesized, annealed into oligo adapters, and sequentially cloned into the entry vector pOs-sgRNA and the Gateway destination vector pOs-Cas921. The CRISPR/Cas9 plasmids were introduced into Agrobacterium tumefaciens strain GV3101 via heat shock. Rice transformation was performed as previously described22. Positive knockout lines were confirmed by PCR followed by sequencing. Hygromycin-sensitive individuals from the correct OsCNGC13 knockout lines in the Nipponbare background (KO-OsCNGC13) were selected, propagated, and used to generate marker-free lines for complementation experiments. For the complementation test, a construct of a 6.9 kb fusion DNA fragment, pLP::etd1-GFP, containing ~ 3.8 kb of the etd1 promoter region, the full etd1 coding sequence, carbon terminal green fluorescent protein (GFP) and the nopaline synthase terminator was built in pCAMBIA1305.2. The plasmid was introduced into Agrobacterium tumefaciens GV3101, which was used for transformation using the hygromycine sensitive KO-OsCNGC13-81 line as the recipient. The derived T2 and T3 progeny were analyzed for all transgenic lines. Transmission electron microscopy observation For TEM analysis, samples of spontaneous cell death and healthy tissues from fully expanded leaves of 3-week-old etd1 plants were fixed in 2.5% glutaraldehyde (pH 7.0) at 4°C for 4 h, followed by post-fixation in 1% OsO4 (pH 7.0) for at 4°C for 12 h. The samples were dehydrated through an ethanol gradient (30%-95%), transitioned to absolute acetone, and embedded in Spurr resin. After polymerization at 70°C for 9 h, ultrathin sections were cut using a LEICA EM UC7 Ultratome, stained with uranyl acetate and alkaline lead citrate, and imaged using a Hitachi H-7650 TEM. Subcellular localization assay As previously described, the subcellular localization of OsCNGC13 and etd1 proteins in rice protoplasts was investigated23. The full-length CDS of OsCNGC13 and etd1 were cloned into the pRTV-GFP vector to generate OsCNGC13-GFP and etd1-GFP fusion proteins. A previously characterized plasma membrane marker, OsRac3 fused with mCherry was used for co-localization analysis24. Both constructs were co- Page 9/18 transformed into rice protoplasts for subcellular localization studies. Images were captured using a confocal laser scanning microscope (Zeiss LSM 880). Phylogenetic analysis The full-length amino acid sequences of CNGC family proteins in rice and Arabidopsis were obtained from the Rice Genome Annotation Project (http://ricegaas.dna.affrc.go.jp/) and The Arabidopsis Information Resource (https://www.arabidopsis.org/), respectively. Protein sequences containing CNGC- like CNBD domains were retrieved from Phytozome (https://phytozome-next.jgi.doe.gov/pz/portal.html). Using the amino acid sequence of OsCNGC13 as a query, the BLASTP program with an Expect (E) threshold of "1e-1" was employed to search the proteome databases of 64 plant species listed in Phytozome 12.1. Multiple sequence alignment of the proteins was performed using Clustal X (2.0), and a phylogenetic tree of the aligned sequences was constructed using the maximum likelihood method in MEGA11. Electrophysiological Studies in Xenopus laevis Oocytes To validate the Ca2+ channel activity of etd1, expression and two-electrode voltage clamp (TEVC) recordings in X. laevis oocytes were performed as previously described25. OsCNGC13-GFP, etd1-GFP, etds102-GFP, etds145-GFP and etds176-GFP were cloned into the pGEMHE vector. Capped RNAs (cRNAs) of genes to be tested were transcribed in vitro from linearized pGEMHE vectors using the mMESSAGE mMACHINE T7 kit (Ambion). Stage V-VI X. laevis oocytes were harvested and stored overnight in ND96 perfusion solution (96 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5 mM HEPES, 10 mM sorbitol, pH 7.4 adjusted with NaOH) before injection. Each oocyte was injected with 25 ng of cRNAs or an equivalent volume of water. Injected oocytes were incubated in perfusion solution at 18°C for two days, and protein expression was confirmed by GFP fluorescence (excitation at 488 nm) using a Zeiss LSM 880 confocal microscope. Currents were recorded using an Axon Axoclamp 900A microelectrode amplifier with a hyperpolarizing pulse of -40 mV for 0.1 s as a pre-pulse, followed by voltage steps from 0 to -180 mV (-20 mV increments, 1.5 s duration) and a -40mV deactivation step for 0.5 s. The Ca2+ bath solution for current recordings contained 1mM, 10 mM or 30 mM CaCl2, 10 mM MES-Tris (pH 7.5) and was adjusted to 220 mOsm/L with mannitol. The pipette solution contained 3 M KCl. For ion selectivity analysis in the etd1, K+ bath solution contained 100 mM KCl, 1 mM MgCl2, 10 mM MES-Tris (pH7.5); ND96 was used for Na + bath solution. The voltage dependence of inward rectification current was fitted with a Boltzmann function26: I k = Imax /{1 + exp[zF / RT (V – V1/2 )]} , where Ik is current at each membrane potential (V), Imax is the unblocked current, V1/2 is the membrane potential where 50% of the channels are blocked and z is the effective valency of the block. R, T and F represent the gas constant, absolute temperature and Faraday’s constant, respectively, with their usual values. Page 10/18 Ca 2+ flux assay with scanning ion-selective electrode technique. Non-invasive micro-test technology (NMT-YG-100, Younger USA) was used to measure ion flux27. Seedlings of IR64 and the etd1 mutant plants were grown in a growth chamber (12 h light cycle, 28°C) on ½ MS medium for approximately 2 weeks. Prior to measurement, seedling stems were cut into 1 cm segments and incubated overnight in a test buffer (0.1 mM KCl, 0.1 mM CaCl2, 0.1 mM MgCl2, 0.5 mM NaCl, 0.3 mM MES, 0.2 mM Na2SO4, pH 6.0). For stem segment measurements, baseline readings were recorded continuously at room temperature until stabilization. Subsequently, CaCl2, chitin, or rice blast elicitors were gradually added to the measurement buffer to achieve final concentrations of 30 mM CaCl2, 10µM chitin, or 1% (v/v) rice blast elicitors. Ca2+ influx was recorded every 6 seconds. Data were analyzed using Excel to calculate specific ion influx (pmol cm− 2 s− 1). Maximum Ca2+ influx was quantified to assess triggered Ca2+ influx. Cameleon technique to monitor Ca 2+ oscillation. Transgenic IR64 and etd1 lines stably expressing the YC3.6 Ca²⁺ biosensor was used to monitor Ca²⁺ oscillations, as described previously28. Sheath tissues from rice seedlings grown under chamber conditions (dark culture, 28°C) on ½ MS medium (to avoid chloroplast autofluorescence) were excised and placed in a 4 mm bottom well dish (Cellvis). To maintain moisture, 100 µl of ½ MS buffer was added. After 1 minute of baseline measurement at room temperature, CaCl2, chitin, or elicitor extract was added to the measurement buffer to achieve final concentrations of 30 mM, 10 µM, or 10% (v/v), respectively, followed by immediate Ca²⁺ imaging. Images were acquired using a Zeiss LSM 880 with a 458 nm laser at 100% power. Emission spectra were recorded at 465–505 nm for ECFP and 520–570 nm for cpVenus, with images captured simultaneously at 3-second intervals. The average fluorescence intensities of the cpVenus and ECFP channels were recorded, and the cpVenus /ECFP ratio over time was plotted to quantify trend of Ca²⁺ changes. Ratio images were generated using ImageJ. Ca 2+ imaging under infection conditions To investigate the intracellular Ca²⁺ accumulation at M.oryzae infection sites, sheath tissues at various infection time points were stained using a Ca²⁺ affinity fluorescent probe. CA89 spores were adjusted to a concentration of 1×10⁵ spores/mL and injected into rice stems using a 1mL syringe. The inoculated stems were placed in 10cm × 10 cm germination boxes lined with moist filter paper to maintain high humidity, followed by initial incubation at 25°C in the dark for 24 hours and subsequent transfer to a growth chamber at 28°C for disease development. For Fluo-4/AM staining, stems at different infection stages were immersed in 10 mM MES buffer or 10 mM MES buffer containing 20 µM Fluo-4/AM (40704ES72, YESEN) and incubated in the dark at room temperature for 5 hours. Prior to microscopic analysis, samples were washed with fresh MES buffer devoid of Fluo-4/AM. Fluorescence imaging of Fluo-4/AM-loaded stem tissues was conducted using a laser scanning confocal microscope (Zeiss LSM 880) with excitation at 488 nm and emission detection at 520 nm. Page 11/18 Protein modeling For homology modeling, the cyclic GMP-activated channel TAX-4 from Caenorhabditis elegans (PDB: 5H3O) was used as a template to model the topologies of CNGC13, etd1 and their core channel domain29. Homology modeling was performed using the SWISS-MODEL online service (https://swissmodel.expasy.org/interactive#structure). For structural prediction of OsCNGC13 and etd1 homotetramers, AlphaFold2 Multimer was employed through ColabFold (v1.5.2)30. (https://colab.research.google.com/github/sokrypton/ColabFold/blob/main/AlphaFold3.ipynb). Four copies of the 735-residue sequences were submitted, and 20 refinement cycles were performed for each of five independent models to enhance prediction accuracy. All PDB data were visualized and processed in the PyMol Molecular Graphics System. Quantification and Statistical Analysis Quantitative analysis of lesion and cell death areas, Ca2+ channel activity, Ca2+ flux, Ca2+ accumulation, grain yield, and other measured parameters was performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA) and PASW Statistics 26. All values are expressed as mean ± standard deviation (SD) or standard error (SE), with the exact value of n (sample size) indicated in the figures or figure legends. Statistical significance of differences was assessed using Student's t-test and Duncan's new multiple range test. Detailed descriptions of the quantification and statistical analysis can be found in the corresponding figures, figure legends, or methods sections. Declarations Funding The study was supported by the National Natural Science Foundation of China (U20A2021, 32201884) and National Key Research and Development Program of China (2023YFF1001200). H.L. was supported by the China Postdoctoral Science Foundation (GZC20230908, 2023M741289). Acknowledgements We would like to thank Drs. Daoxin Xie (Tsinghua University, China), Chuanqing Sun (China Agricultural University, China)  and Yunkai Jin (Hunan Agricultural University, China) for critical reading and discussion. Dr. Lizhong Xiong (Huazhong Agricultural University) for providing YC3.6 vectors and technical support. Dr. Qinlong Zhu (South China Agricultural University) for providing a set of rice subcellular localization marker vectors. Dr. Yuese Ning (Institute of Plant  Protection, Chinese Academy of Agricultural Sciences) for providing a set of rice genetic transformation vectors. Author contributions Page 12/18 J.W., W.S., and B.Z. conceived the project. J.L., H.L., G.X., B.B., X.L., J.Y., S.L., F.Y. performed the experiments and analyzed the data. G.X., J.L., Y.L., B.Z. developed materials. J.L., B.Z., and J.W. wrote the paper. 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Nat Methods 19:679–682 Figures Page 14/18 Figure 1 etd1 regulates both spontaneous and elicitors-triggered cell death and confers broad-spectrum resistance to rice blast. a-b, Phenotypes of youngest fully expanded leaves at different ages in the etd1 mutant and wild-type, IR64 (a), etd1(b). c, The degree of cell death per unit leaf area in the etd1 mutant positively correlates with development stages. Linear regression analysis using Pearson correlation coefficient (R > 0.75, p < 0.05). d, Ultrastructural comparison of cytoplasts between healthy and cell death regions in etd1 leaves. I-III, Healthy regions: (I) a healthy cell, (II) intact nucleus, (III) plump chloroplasts. IV-IX, Cell death regions: (IV) hypersensitive response-like cell, (V) nuclear membrane blebbing and degradation, (VI) ruptured chloroplast membranes, (VII) cell corpses, (VIII) plasmolysis and membrane dissolution, (IX) plasma membrane blebbing. n, nucleus; c, chloroplast; g, grana stack; v, vacuole; pl, plasmodesmata; m, mitochondria; pm, plasma membrane; cw, cell wall; b, blebbing of nuclear or plasma membrane; l, membrane lysis. e-f, etd1 leaves exhibits cell death phenotype induced by M. oryzae. Inoculation with IR64-avirulent isolate V86010 (e). Inoculation with IR64-virulent isolate Page 15/18 CA89 (f). CO39 was used as a susceptible control. g, Cell death phenotypes in etd1 induced by chitin or rice blast elicitors. h, Cell death per unit leaf area caused by different treatments. i, etd1 conferred broad- spectrum resistance to diverse rice blast isolates. The leaves are shown 5 days after inoculation with eight IR64-virulent isolates (out of a total of 106 isolates tested), complete data are available in Supplementary Table 1. j, Disease symptoms and cell death in leaves. k, Microscopic images of sheath cells from IR64 and etd1 at different time points post-infection with CA89. l, Distribution of fungal infection at 12, 24, 36, and 48 hpi. At least 100 single-cell interaction sites were examined by replicate. Bars represent the meaning of three replicates. m, Structure of the etd1 gene and the mutation in the etd1 mutant. Black filled boxes represent exons of Os06g10580. The etd1 mutant carries a SNP in exon 7, nucleotide and deduced amino acid sequences of exon 7 in IR64 and the etd1 mutant are shown below. A G-to-A substitution (highlighted in red) that changes glycine to glutamic acid at position 483. n, The leaf phenotypes at the flowering stage of etd1 genetic complementation-related lines. Two independent OsCNGC13 knockout lines in the NIP background showed no cell death phenotypes. In contrast, two pLP::etd1-GFP (genetic complementation constructs of etd1) transgenic lines in the KO- OsCNGC13 (marker-free) background exhibited spontaneous cell death phenotypes. o-p, Phenotypes after challenge with M. oryzae of the etd1 genetic complementation-related lines. The KO- OsCNGC13lines showed rice blast resistance identical to NIP, while the pLP::etd1-GFP transgenic lines exhibited etd1-like elicitors-triggered cell death phenotypes. Inoculation with NIP-avirulent isolates 4029- 1 (o). Inoculation with NIP-virulent isolate S5 (p). In c, h, j, l, data are mean ± s.d.; n = 3, biologically independent samples. In c,h, the same lowercase letters indicate no significant difference at P > 0.05 as determined by two-way ANOVA with LSD test. Scale bars, 5 mm (a), 50 µm (k). Page 16/18 Figure 2 The non-canonical rectifying Ca2+ channel etd1 mediates robust Ca2+ influx triggering cell death. a, 3D structural comparison of OsCNGC13 and etd1 generated using Swiss-Model (template: PDB: 5H3O). Q486 represents the gating residue of the CNGC channel. b, etd1 exhibits enhanced Ca²⁺-permeable channel activity compared to OsCNGC13 in X. laevis oocytes. Current-voltage relationships were recorded in oocytes expressing OsCNGC13-GFP, etd1-GFP or co-expressing both in the presence of 30 Page 17/18 mM CaCl₂. Voltage steps of 0 to -180 mV in 20 mV decrements are shown. The I-V curves of oocytes expressing OsCNGC13 exhibit a nonlinear relationship (classic rectifying property), with the gray arrow indicating that, when the voltage reaches -160 mV, the Ca2+ influx current begins to increase significantly relative to the mock with increasing voltage. Expressing etd1 show a linear relationship (a non-classic rectifying property), with the dark arrow indicating that, when the voltage reaches -20 mV, the Ca2+ influx current starts to increase significantly compared to the mock as the voltage rises. Number of biological replicates: control (water-injected, n = 6), OsCNGC13 (n =8), etd1 (n =12) and  OsCNGC13+etd1 (n =14). c, Boltzmann method for fitting I/Imax-V curves (top) and half-voltage (V1/2) and effective valency (z) of the inward rectification. Higher V1/2 and z value indicate stronger rectification. (d, h) Non-invasive micro- test (NMT) measurements of extracellular Ca²⁺ influx in living cells of IR64 and etd1 stems. After 5 minutes of baseline recording under normal conditions, seedlings were measured for 6 minutes in test buffer. Treatments: 30 mM CaCl₂ (d) or 10 µM chitin (h). (e, i) Quantification of maximum Ca2+ influx determined by NMT. Treatments: 30 mM CaCl₂ (e) or 10 µM chitin (i). (f, j) The time-course dynamics of cytosolic Ca2+ elevation in living stem from the IR64 and etd1 expressing Yellow Cameleon (YC3.6). The cytosolic Ca2+ elevation was evaluated using the normalized ratio of cpVenus/ECFP (enhanced cyan fluorescence protein). Treatments: 30 mM CaCl₂ (f) or 10 µM chitin (j). (g, k) Quantification of peak ratio (cpVenus/ECFP) using YC3.6. Treatments: 30 mM CaCl₂ (g) or 10 µM chitin (k). l, Representative laser scanning microscopy images of IR64 and etd1 sheath cells stained with Fluo-4 at 12, 24, 36, and 48 hpi challenge with CA89. At least 20 single-cell interaction sites were examined by replicate. Fluorescence intensity per unit area was used to quantify intracellular Ca²⁺ accumulation. White triangles indicate M. oryzae infection sites. m, Phenotypes of 3-week-old plants hydroponically cultured with 0.01, 0.1, 0.3, 0.5, or 1 mM CaCl₂. n, The ratio of cell death per unit leaf area in etd1 exhibits a positive correlation with the concentration of [Ca2+]ext. o, The etd1 triggered cell death phenotype is calcium-dose dependent. IR64 and etd1 were cultured for 3 weeks under different Ca2+ conditions inoculated with CA89 and leaf phenotypes were analyzed 5 days later. Black triangles indicate typical rice blast lesions in etd1 leaves under 0.01 mM Ca2+ conditions. p, Location of amino acid modifiers in the etd1 resulting from mutagenesis. The positions of three independent mutations and their corresponding amino acid changes (1-3) are marked by green arrows, displayed using PROTTER (https://wlab.ethz.ch/protter/start/) to indicate transmembrane and exposed regions. Residues distinguishing OsCNGC13 from etd1 are highlighted in red arrow. q-t, The etds mutants completely suppressed cell death phenotype induced by M. oryzae and elicitors. Treatments: inoculation with V86010 (q), inoculation with CA89 (r), mock treatment (s), rice blast elicitors treatment (t). u, Current- voltage relationships were recorded in X. laevis oocytes expressing OsCNGC13, etd1 and different etds in the presence of 30mM CaCl₂, with voltage steps from 0 to -180 mV in 20 mV decrements. Controls (n = 5), OsCNGC13, etd1 and etds (n=8). v, Quantification of current amplitudes at -180 mV under 30 mM CaCl₂. In b, u data are mean ± s.e.; c-k, l, n, o, v, data are mean ± s.d.. In c, n, o, v, the same lowercase letters indicate no significant difference at P > 0.05 as determined by two-way ANOVA with LSD test. In e, g, i, k, l, the Student’s t-test analysis indicates a significant difference (**P < 0.01). In n, o, linear Page 18/18 regression analysis using Pearson correlation coefficient (R > 0.75, p < 0.05). Scale bars, 50 µm (l), 2 cm (m). Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. SupplementalInfo.docx SupplementaryofNGBC68301.docx https://assets-eu.researchsquare.com/files/rs-6155589/v1/34c267ec8d12409e5ed94abd.docx https://assets-eu.researchsquare.com/files/rs-6155589/v1/e9c9e9042d55cf0dd4785fa9.docx