SPL50 negatively regulates cell death and disease resistance in rice

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Based on ethyl methane sulfonate (EMS)-induced mutagenesis, the spl50 mutant mimics hypersensitive responses in the absence of pathogen by displaying spontaneous necrotic lesions after the tillering phase. SPL50, an ARM repeat protein essential for controlling reactive oxygen species (ROS) metabolism and boosting resistance to blast disease, was identified by map-based cloning techniques. This work also demonstrates the detrimental effects of spl50 on photosynthetic efficiency and chloroplast development. The crucial significance of SPL50 in cellular signaling and stress response is shown by its localization to the cell membrane and constitutive expression in various plant tissues. Given increasing concerns about global food security, this research underscores the critical role of SPL50 in modulating PCD and fortifying the immune response, contributing to the development of strategies for enhancing crop disease resistance. SPL50 ARM repeat protein cell death disease resistance rice Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Introduction Plant lesion mimics (LMMs) are a remarkable phenomenon in plant biology that reveal how plants can exhibit symptoms even when they are not experiencing disease or environmental stress (Zhu et al., 2020 ). These necrotic spots, varying in shapes and sizes, emerge spontaneously across various plant parts, including the leaves and leaf sheaths. The fact that this phenomenon develops independently of biotic (pathogen-related) or abiotic (environmental stress-related) factors raises the possibility that an underlying genetic or epigenetic mechanism is at work. Numerous plant species exhibit lesion mimic mutants, underscoring a widespread genetic foundation for this phenomenon. Examples include barley (Wolter et al., 1993 ), rice (Takahashi et al., 1999 ), Arabidopsis (Lorrain et al., 2003 ), maize (Mu et al., 2021 ), and wheat (Yu et al., 2023 ; Wang et al., 2023), among others. These mutant`s lesions bear a striking resemblance to those seen in plants after a hypersensitive response (HR), a well-documented form of programmed cell death (PCD) (Cai et al., 2021 ). The HR plays a crucial role in controlling the spread of diseases by rapidly eliminating contaminated cells and enclosing the afflicted area to stop the germs from moving further. A key component of the plant's innate immune system, this fast cell death mechanism allows it to successfully fend off pathogen attacks without the involvement of adaptive immunity system, which are present in more complex species. To date, an array of LMM genes has been elucidated, encoding proteins that span a diverse range of functions. These include the MEDIATOR SUBUNIT 16 (Zhang et al., 2023 ), the deubiquitinase OsLMP1 (Zou et al., 2023 ; Sun et al., 2022), receptor-like protein kinase SPL36 (Rao et al., 2021 ), translation factor OsWRKY19 (Du et al., 2021 ), ATP-citrate lyase SPL30 (Ruan et al., 2019 ), eukaryotic translation elongation factor 1 alpha (eEF1A)-like protein SPL33 (Wang et al., 2017 ), splicing factor 3b subunit SPL5 (Chen et al., 2012 ), and heat shock transcription factor SPL7 (Yamanouchi et al., 2003). Each of these genes plays a distinct and pivotal role in the intricate biological phenomena observed in plants, especially in underlining the mechanisms of disease resistance and PCD in rice. The discovery of Armadillo (ARM) repeat proteins, initially identified in Drosophila melanogaster , signified a noteworthy progression in our comprehension of protein structure and function. These proteins are characterized by a highly conserved structural domain consisting of triplets of α-helices, each arranged in a repeating 42-amino acid pattern (Peifer et al., 1994 ). This unique structural design contributes to their adaptability and allows ARM repeat proteins to assume various conformations, adopting to different functional roles as required. The adaptability of ARM repeat proteins is a crucial aspect of their ability to interact with a wide range of protein partners. This flexibility facilitates their involvement in numerous essential cellular processes, underscoring their importance in maintaining cellular homeostasis and responding to internal and external signals. One critical role of ARM repeat proteins is in nucleo-cytoplasmic transport, which is fundamental to the proper functioning and regulation of cells (Wang et al., 2014 ). This process ensures that substances necessary for cellular function are correctly shuttled between the nucleus and the cytoplasm, allowing for the appropriate expression and regulation of genes. Additionally, ARM repeat proteins are vital in mediating cellular responses to environmental cues through signal transduction pathways (Kulich et al., 2020 ). These pathways are essential for the cell's ability to detect and respond to changes in its environment, ensuring cellular adaptation and survival under various conditions. protein trafficking is another crucial process involving ARM repeat proteins (Yoshida et al., 2023 ). This mechanism is responsible for the correct transport and localization of proteins within the cell, ensuring that proteins delivered to where they are needed mos. Furthermore, ARM repeat proteins play a significant role in the ubiquitination processes, which tags proteins for degradation or functional modification (Wang et al., 2023; Lv et al., 2022 ). ARM repeat proteins are pivotal across a vast array of organisms, playing particularly significant roles in plant biology where they regulate development, stress responses, and metabolic pathways. In Arabidopsis thaliana , the ARM domain-containing protein ARK1 interacts directly with RHD3 to modulate the architecture of the endoplasmic reticulum, a key factor in maintaining cellular integrity and function (Sun et al., 2020 ). This interaction underscores how ARM repeat proteins facilitate the organization of cellular structures. In terms of stress adaptation, ARM repeat proteins like OsPUB2 and its homolog OsPUB3 in rice significantly enhance the plant's tolerance to cold stress, reflecting the functional adaptability of these proteins in response to environmental challenges (Byun et al., 2017 ). Additionally, ARM repeat proteins play integral roles in plant defense mechanisms. For example, OsIMα1a and OsIMα1b are crucial in defending rice against blast disease, thereby bolstering crop resilience and food security (Xu et al., 2022 ). The interaction of these proteins with pathogenic challenges illustrates the dynamic role of ARM repeat proteins in mobilizing plant defenses against external threats. Adding to the complexity, proteins like SPL11 and OsPUB15, which possess both ARM and U-box domains, are pivotal in the ubiquitin-mediated regulation of disease resistance mechanisms within rice (Zeng et al., 2004 ; Wang et al., 2015 ). Despite extensive research on ARM repeat proteins within the plant kingdom and their proven significance in multiple biological functions, the roles of many members within the ARM family remain largely unexplored. This gap in our knowledge underscores the necessity for ongoing research efforts to elucidate the complex mechanisms through which ARM repeat proteins influence cell death, disease resistance, and other critical processes. Unraveling these mechanisms is essential not only to advance our fundamental understanding of plant biology but also to exploit this knowledge in developing crops with enhanced resistance to stresses and diseases. Such research is crucial for promoting sustainable agriculture and ensuring food security amidst escalating global challenges. To investigate the molecular mechanisms governing cell death and disease resistance in rice, we successfully isolated a novel LMM, named spotted leaf 50 ( spl50 ), from ethyl methane sulfonate (EMS)-mutagenized Oryza sativa japonica cv. Wuyunjing 7 (WYJ). The spl50 mutant displayed distinct spotted leaves beginning at the tillering stage and continuing through the ripening phase, indicative of internal disruptions potentially linked to innate immunity mechanisms. To identify the genetic basis of the spl50 phenotype, we employed a map-based cloning strategy. This approach led us to discover that the mutation responsible for the spl50 characteristics occurred in a gene encoding an ARM repeat protein. ARM repeat proteins are known for their role in various cellular processes, including developmental regulation and stress response. The mutation in the SPL50 gene led to a notable accumulation of reactive oxygen species (ROS) in the mutant plants. Interestingly, despite the potential for damage, the increased ROS levels in spl50 mutants were also associated with enhanced resistance to blast disease. This observation suggests that SPL50 plays a dual role in maintaining ROS homeostasis and activating defense mechanisms against pathogens. This finding adds a new layer to our understanding of SPL50's functions, positioning it as a crucial player in the plant’s innate immune response.. Results Identification of the spl50 mutant The discovery of the spl50 mutant resulted from a meticulous screening process subsequent to the application of EMS (ethyl methane sulfonate) mutagenesis on the japonica rice cultivar, Wuyunjing 7 (WYJ). At the seedling stage, the spl50 mutant under field conditions showed no noticeable differences from the wild type, including the absence of visible lesions on its leaves (Fig. 1 A-B). As the plants matured from the tillering to the ripening stages, however, pronounced phenotypic differences began to emerge. Both aging and newly formed leaves of the spl50 mutants developed reddish-brown lesions, contrasting sharply with the vibrant green foliage observed in wild type plants (Fig. 1 C-F). This shift in leaf coloration and the appearance of lesions are indicative of significant internal changes within the plant, potentially signaling a hypersensitive response or a self-initiated activation of cell death mechanisms, occuring in the absence of pathogen interaction. Such phenotypic changes suggest that the spl50 mutation may have profound effects on the plant’s vitality and stress response pathways. Moreover, the spl50 mutants exhibited considerable reductions in several agronomic traits compared to their wild type counterparts, such as plant height, grain size, 1000-grain weight, the number of productive panicles, the number of grains per panicle, primary and secondary panicle branches, grain yield per plant, total grain number per plant, and seed setting rate (Fig. S1 ). Alterations in reactive oxygen species (ROS) metabolism in the spl50 mutant The appearance of lesion-mimic spots on the leaves of spl50 mutants serves as a visible indicator of the activation of cellular death pathways, accompanied by a notable accumulation of hydrogen peroxide (H 2 O 2 ) within the cells. To confirm this phenomenon, we conducted a series of experiments utilizing 3,3'-diaminobenzidine (DAB) staining on both wild type and spl50 mutant leaves over an 8-hour period. The results revealed pronounced brown pigmentation exclusively in spl50 leaves, indicative of a significant buildup of H 2 O 2 , contrasting sharply with the unaltered appearance of wild type leaves (Fig. 2 A). Furthermore, Trypan blue staining highlighted extensive cellular damage within spl50 leaves, diverging markedly from the unaffected wild type (Fig. 2 B). Quantitative analyses further supported these observations, demonstrating elevated levels of H 2 O 2 and superoxide anion (O 2− ) within the spl50 mutant, along with increased production of malondialdehyde (MDA), a marker signifying both cellular demise and lipid peroxidation (Fig. 2 D-F). Enzymatic analyses revealed a decrement in catalase (CAT) activity within spl50 , whereas peroxidase (POD) and superoxide dismutase (SOD) activities experienced significant elevation, underscoring an intensified oxidative stress response in the mutant (Fig. 2 G-I). Concomitantly, transcriptomic analyses unveiled a significant upregulation of five senescence-associated genes, namely WRKY23 , SGR , OsI85 , Osh69 , and RCCR1 , in the spl50 mutant (Fig. 2 C). This finding affirms the genetic response to oxidative stress and cellular senescence in spl50 . To further investigate the cellular death mechanisms in spl50 , we examined chromatin condensation within mature leaf cells. the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay can be used to identify the endonucleolytic fragmentation of nuclear DNA, which is the mechanism that is unmistakably associated with programmed cell death (PCD). This analysis unveiled a distinct disparity between the spl50 mutants and their wild type counterparts. Specifically, the spl50 mutants have a high frequency of TUNEL-positive nuclei, whereas the wild type has a very low frequency of these nuclei (Fig. 3 ). Enhanced innate immune responses in the spl50 mutant Magnaporthe oryzae , the fungus that causes rice blast disease, has caused a drastic decline in cereal yields over 30%, posing a significantly threat to global food security (Zhang et al., 2024 ). In light of this pressing issue, our study sought to assess on evaluating the resistance of the spl50 mutant against this formidable pathogen. After two weeks, specimens of both wild type and spl50 mutant plants, were inoculated with the M. oryzae strain R01-1. A week after inoculation, careful monitoring of disease symptoms was undertaken. Significantly, the spl50 mutant leaves showed noticeably less lesions than the wild type leaves, which displayed extensive necrotic lesions (Fig. 4 A-B). To further substantiate the extent of fungal infiltration and proliferation within the leaf tissues, DNA was meticulously extracted from matched samples of both wild type and spl50 mutant leaves. The subsequent quantification of M. oryzae biomass using quantitative real-time PCR (qRT-PCR) unveiled a significantly diminished fungal biomass in the spl50 mutant compared to the wild type (Fig. 4 C). Additionally, a comprehensive quantitative analysis of the expression levels of key defense genes, including PR1a , PR1b , PR10 , PBZ1 , and SL , was conducted via qRT-PCR. The results revealed a substantial upregulation of these genes in the spl50 mutant (Fig. 4 D). This robust induction of these defense genes in the spl50 mutant provides compelling evidence for its enhanced resistance to the rice blast disease. Our findings underscore the potential of the spl50 mutation in bolstering the plant's defense mechanisms against M. oryzae . Impact of SPL50 mutation on chloroplast development and photosynthetic efficiency Lesion mimic phenomena are frequently associated with disruptions to chloroplast ontogeny, aberrant gene expression patterns related to chloroplast, and reduced levels of chlorophyll, all of which have a negative impact on photosynthetic efficiency (Cui et al., 2021 ; Wang et al., 2017 ). In an effort to determine if the spl50 mutant exhibits similar perturbations, extensive investigations were carried out on heading-stage wild type and spl50 plants. This involved quantifying the amount of chlorophyll and assessing the expression of protein linked to chloroplasts in their leaves. As compared to the wild type, our findings showed a considerable drop in the levels of chlorophyll a, chlorophyll b, and carotenoids in the spl50 mutant indicating a major impairment in chlorophyll biosynthesis and accumulation (Fig. 5 A). Further, the spl50 mutation has a significant influence on the integrity and function of chloroplasts, as demonstrated by the increased degradation of several key proteins associated to chloroplast, including Tic110, CP47, RbcL, D1, and PsbO, as revealed by western blot analysis (Fig. 5 B). Subsequent examination of the photosynthetic efficiency within the flag leaves of both genotypes at the heading stage unveiled a marked decline in the net photosynthetic rate, stomatal conductance, and transpiration rate in the spl50 mutant (Fig. 5 C). This reduction was accompanied by an elevated intracellular CO 2 concentration, indicating a severe disruption in the photosynthetic machinery. These observations provide further evidence that the spl50 mutation adversely affects chloroplast functionality in rice, leading to a pronounced decrease in photosynthetic efficiency. Map-based cloning of SPL50 To conduct a genetic analysis of the spl50 mutant, we initiated a cross between the mutant phenotype and the wild type cultivar WYJ. Phenotypic examination of all progeny in the F 1 generation revealed wild type characteristics, indicating the recessive nature of the mutation. In the F 2 population, comprising 441 plants, 338 exhibited growth patterns similar to the wild type, while the remaining displayed the mutant phenotype. The observed segregation ratio of 3:1 ( χ 2 0.05 = 0.477 < 3.841) substantiates the hypothesis that the spl50 mutation is governed by a single recessive allele at a nuclear locus. For the purpose of identifying the gene associated with the mutant phenotype, the spl50 mutant was crossed with an indica rice variety 9311. From this cross, 1345 F 2 progeny manifesting the mutant phenotype were selected for further analysis. We employed a total of 183 simple sequence repeats (SSRs) and 41 sequence tagged sites (STSs), which are uniformly dispersed across the 12 chromosomes of rice, for genotyping. Initial mapping efforts positioned the mutant locus between markers B10-1 and B10-2 on chromosome 10, as determined by analyzing the 113 F 2 plants (Fig. 6 A). Subsequent fine mapping, utilizing a larger set of 1232 F 2 mutants, refined the locus to a 112-kb interval flanked by markers P7 and P8 (Fig. 6 B-C). According to data from the Rice Genome Annotation Project (RGAP, http://rice.plantbiology.msu.edu ), this interval contains sixteen predicted open reading frames (ORFs). Sequencing of this specific region in the spl50 mutant identified a transversion mutation from T to A within an exon of LOC_Os10g05370 , resulting in a Leu-to-His substitution at the 343th amino acid (Fig. 6 D-G). Thus, LOC_Os10g05370 has been identified as the candidate gene responsible for the observed mutant phenotype. Confirmation of LOC_Os10g05370 responsible for the mutant phenotype of spl50 To definitively confirm that SPL50 corresponds to LOC_Os10g05370 , we meticulously inserted a 5.1 kb genomic fragment of LOC_Os10g05370 into the binary vector pCAMBIA1300, followed by its introduction into the spl50 mutant via transformation. This process yielded 23 independent T 0 transformants. Detailed phenotypic analysis clearly demonstrated that each transformant effectively mitigated the spl50 mutant phenotype, as evidenced by an enhanced plant height and the elimination of lesion-mimic spots (Fig. 6 H). Subsequent exposure of WT, spl50 , and the complemented line p GSPL50-1 to the rice blast pathogen underscored the functional restoration of disease resistance. Notably, there were no significant differences in the extent of lesion formation and pathogen biomass accumulation between p GSPL50-1 and WYJ (Fig. 7 A-C). In addition, a pronounced reduction in the expression levels of defense-related genes OsPR1a and OsPR1b in p GSPL50-1 compared to spl50 was observed, indicating a normalization of the defense response (Fig. 7 D-E). These findings provide compelling evidence supporting the hypothesis that the lesion mimic phenotype observed in the spl50 mutant is attributable to a single-base substitution mutation within the LOC_Os10g05370 gene of SPL50 . Constitutive expression of SPL50 Through qRT-PCR analysis, our investigation revealed the broad expression spectrum of SPL50 gene across diverse plant tissues, such as roots, culms, leaves, leaf sheaths, and panicles. Notably, SPL50 exhibited its highest expression level of in leaf tissue, while its lowest expression was observed within the panicle structures (Fig. 8 A). To further elucidate the regulatory elements governing SPL50 expression, we engineered a construct by fusing a 2,052-bp genomic fragment, located upstream of SPL50's start codon, with the β-glucuronidase (GUS) reporter gene. Subsequently, this recombinant DNA was introduced into the genome of wild type plants through transformation. Histochemical analysis of GUS activity in the progeny of these transgenic plants revealed enzymatic activity in roots, culms, leaves, and panicles (Fig. 8 B). This pattern of GUS expression closely mirrors the expression profile of SPL50 as determined by qRT-PCR, thus corroborating the constitutive expression paradigm of SPL50 across the examined plant tissues. Subcellular localization of SPL50 To elucidate the subcellular localization of SPL50, our study employed a strategic approach involving the creation of a recombinant expression vector (p35S::SPL50-GFP). This vector was ingeniously designed to fuse the SPL50 coding sequence with the green fluorescent protein (GFP) marker, enabling visual tracking of the protein within cells. Subsequently, this construct was introduced into the cellular environment of rice protoplasts and leaf epidermal cells of tobacco ( Nicotiana benthamiana ), serving as a model system for plant cell biology studies. Utilizing advanced confocal microscopy techniques, we meticulously observed the distribution of the SPL50-GFP fusion protein within these cells. Our findings compellingly demonstrate that SPL50 predominantly localizes to the cell membrane (Fig. 9 ). This localization pattern suggests a potential role for SPL50 in membrane-associated processes or signaling pathways. Discussion The novel identification and characterization of the SPL50 gene within Oryza sativa not only enriches our understanding of plant LMMs but also sheds light on the intricate regulatory networks governing PCD and innate immune responses in plants. SPL50 , encoding an ARM repeat protein, emerges as a pivotal regulator orchestrating PCD and enhancing innate immune responses in rice. The spl50 mutant's spontaneous development of necrotic lesions, independent of pathogen attack, underscores a self-activated PCD pathway, akin to an autoimmune response. This phenomenon is reflective of the mutant's altered ROS metabolism, characterized by excessive accumulation of H 2 O 2 and O 2− (Fig. 2 ), hallmark indicators of oxidative stress and cell death (Mittler, 2002 ). Furthermore, the enhanced resistance of the spl50 mutant to rice blast disease (Fig. 4 ), underscores SPL50 's significant contribution to the plant's immune defense. The observed upregulation of defense-related genes (e.g., PR1a , PR1b , PR10 , PBZ1 , and SL ) (Fig. 4 D) in the spl50 mutant suggests that SPL50 influences the transcriptional regulation of key components in rice's defense signaling pathways. The mechanisms by which SPL50 regulates PCD and immune response likely involve its ARM repeat domains, facilitating protein-protein interactions crucial for various cellular processes, including signal transduction, gene expression regulation, and multiprotein complex assembly (Yoshida et al., 2023 ; Wang et al., 2023; Lv et al., 2022 ; Kulich et al., 2020 ; Wang et al., 2014 ). In this study, it is plausible that its ARM domains interact with specific proteins involved in ROS metabolism and defense signaling pathways. The accumulation of ROS in the spl50 mutant (Fig. 2 ) may be attributed to SPL50 's potential role in modulating antioxidant enzyme activities, such as CAT, POD, and SOD. SPL50 might influence the expression or stability of these enzymes, thereby affecting ROS detoxification and the balance between ROS production and scavenging. SPL50's contribution to enhanced disease resistance could involve the modulation of defense signaling pathways, possibly through interactions with key signal transducers or transcription factors involved in immune response activation. The upregulation of defense-related genes in the spl50 mutant (Fig. 4 D) suggests that SPL50 may influence the transcriptional machinery directly or indirectly, perhaps by affecting the stability or activity of transcription factors, which play a central role in plant immune responses. Moreover, the SPL50-mediated regulation of chloroplast functionality and photosynthetic efficiency (Fig. 5 ) indicates a broader impact of SPL50 on plant physiology, connecting PCD and immune response to photosynthetic performance. This relationship underscores the interconnectedness of plant defense mechanisms with other physiological processes, highlighting the complexity of plant responses to environmental stresses. In summary, the SPL50 gene exemplifies the sophisticated regulatory mechanisms plants employ to mediate PCD and innate immune responses. Through its ARM repeat domains, SPL50 likely interacts with a spectrum of proteins to modulate ROS metabolism and defense signaling pathways, playing a crucial role in rice's ability to counteract pathogenic attacks while managing cellular homeostasis. Future studies should aim to elucidate the specific protein-protein interactions involving SPL50, further clarifying its role in plant PCD and immunity. Understanding these mechanisms not only contributes to our fundamental knowledge of plant biology but also offers potential avenues for enhancing crop resistance to diseases, a critical goal in the face of global food security. Conclusions The novel identification of the SPL50 gene in rice enriches our understanding of plant lesion mimic phenomena and unveils its pivotal role in regulating PCD and innate immune responses. Encoding an ARM repeat protein, SPL50 orchestrates PCD and enhances immune responses independently of pathogen attack. Its influence on ROS metabolism and defense signaling pathways underscores its significance in rice's defense mechanisms. The upregulation of defense-related genes in the spl50 mutant suggests SPL50 's involvement in transcriptional regulation. Additionally, SPL50's impact on chloroplast functionality and photosynthetic efficiency highlights its broader physiological effects. Through protein-protein interactions mediated by its ARM repeat domains, SPL50 likely modulates ROS metabolism and defense signaling pathways, contributing to both disease resistance and cellular homeostasis. Further research elucidating SPL50's specific interactions and mechanisms promises insights into plant biology and avenues for crop improvement, vital for global food security challenges. Abbreviations LMMs: Plant lesion mimics; HR: Hypersensitive response; PCD: Programmed cell death; eEF1A: Eukaryotic translation elongation factor 1 alpha; ARM: Armadillo; CAT: Catalase; POD: Peroxidase; SOD: Superoxide dismutase; DAB: 3,3'-diaminobenzidine MDA: Malondialdehyde; EMS: Ethyl methane sulfonate; ROS: Reactive oxygen species; qRT-PCR: Quantitative real-time PCR; SSRs: Simple sequence repeats; STSs: Sequence tagged sites; GUS: β-glucuronidase; GFP: Green fluorescent protein; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling; RGAP: Rice Genome Annotation Project. Declarations Acknowledgements Not applicable. Funding This work was supported by “Ten-thousand Talents Plan” of Zhejiang Province (2022R52027 to Y.Y.), the funds from the Natural Science Foundation of Zhejiang (LY23C130001 to B.R.) and the National Natural Science Foundation of China (32370328 to L.W.). Authors’ Contributions Y. Y, Q. Q, and B. R designed the experiments; B. R, H. W, Y. J, J. Q, F. 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Xu X, Wang H, Liu J, Han S, Lin M, Guo Z, Chen X (2022) OsWRKY62 and OsWRKY76 Interact with Importin α1s for Negative Regulation of Defensive Responses in Rice Nucleus. Rice 15 (1):12. Yamanouchi U, Yano M, Lin H, Ashikari M, Yamada K (2002) A rice spotted leaf gene, Spl7 , encodes a heat stress transcription factor protein. Proc Natl Acad Sci U S A 99 (11):7530-7535. Yoshida MW, Hakozaki M, Goshima G (2023) Armadillo repeat-containing kinesin represents the versatile plus-end-directed transporter in Physcomitrella . Nat Plants 9 (5):733-748. Yu G, Matny O, Gourdoupis S, Rayapuram N, Aljedaani FR, Wang YL, Nürnberger T, Johnson R, Crean EE, Saur IM, Gardener C, Yue Y, Kangara N, Steuernagel B, Hayta S, Smedley M, Harwood W, Patpour M, Wu S, Poland J, Jones JDG, Reuber TL, Ronen M, Sharon A, Rouse MN, Xu S, Holušová K, Bartoš J, Molnár I, Karafiátová M, Hirt H, Blilou I, Jaremko Ł, Doležel J, Steffenson BJ, Wulff BBH (2023) The wheat stem rust resistance gene Sr43 encodes an unusual protein kinase. Nat Genet 55 (6):921-926. Zeng LR, Qu S, Bordeos A, Yang C, Baraoidan M, Yan H, Xie Q, Nahm BH, Leung H, Wang GL (2004) Spotted leaf 11 , a negative regulator of plant cell death and defense, encodes a U-box/armadillo repeat protein endowed with E3 ubiquitin ligase activity. Plant Cell 16 (10):2795-2808. Zhang H, Yang J, Liu M, Xu X, Yang L, Liu X, Peng Y, Zhang Z (2024) Early molecular events in the interaction between Magnaporthe oryzae and rice. Phytopathol Res 6(1):9. Zhang P, Ma X, Liu L, Mao C, Hu Y, Yan B, Guo J, Liu X, Shi J, Lee GS, Pan X, Deng Y, Zhang Z, Kang Z, Qiao Y (2023) MEDIATOR SUBUNIT 16 negatively regulates rice immunity by modulating PATHOGENESIS RELATED 3 activity. Plant Physiol 192 (2):1132-1150. Zhao H, Wang X, Jia Y, Minkenberg B, Wheatley M, Fan J, Jia MH, Famoso A, Edwards JD, Wamishe Y, Valent B, Wang GL, Yang Y (2018) The rice blast resistance gene Ptr encodes an atypical protein required for broad-spectrum disease resistance. Nat Commun 9 (1):2039. Zhu X, Ze M, Chern M, Chen X, Wang J (2020) Deciphering rice lesion mimic mutants to understand molecular network governing plant immunity and growth. Rice Sci 27(4): 278-288. Zou T, Li G, Liu M, Liu R, Yang S, Wang K, Lu L, Ye Q, Liu J, Liang J, Deng Q, Wang S, Zhu J, Liang Y, Liu H, Yu X, Sun C, Li P, Li S (2023) A ubiquitin-specific protease functions in regulating cell death and immune responses in rice. Plant Cell Environ 46 (4):1312-1326. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 13 Aug, 2024 Read the published version in Rice → Version 1 posted Editorial decision: Revision requested 21 Jun, 2024 Reviews received at journal 31 May, 2024 Reviews received at journal 22 May, 2024 Reviewers agreed at journal 21 May, 2024 Reviewers agreed at journal 13 May, 2024 Reviewers invited by journal 13 May, 2024 Editor assigned by journal 30 Apr, 2024 Submission checks completed at journal 30 Apr, 2024 First submitted to journal 25 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4326724","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":298064908,"identity":"defd0bac-9731-489b-a6fa-ea08e7065187","order_by":0,"name":"Banpu Ruan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxElEQVRIiWNgGAWjYJACZgYGCTl59sbGhx9I0GJhbNhzuNlYggQtFYkNN9LbBHiIUW4+I/nZ48IdEoyNMx+2MUgw2MnpNhDQInMjzdx45hkJZnbpxLYHBQzJxmYHCGiRkEgwk+Ztk2BjnJ3YbiDBcCBxG2Et6d9AWngYbh4EkURpyQHbIsFwg5FYLTxvyqSBfjEw7EkEBrIBMX5hT98mXbijrn4++/GHDz9U2MkR1AIGjA0wlgExylG1jIJRMApGwSjAAgCHtTy7HuW+kAAAAABJRU5ErkJggg==","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":true,"prefix":"","firstName":"Banpu","middleName":"","lastName":"Ruan","suffix":""},{"id":298064909,"identity":"f7822338-5927-491b-bf50-939d4ee75e8e","order_by":1,"name":"Hui Wu","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Hui","middleName":"","lastName":"Wu","suffix":""},{"id":298064911,"identity":"3136c4d9-8da5-4890-b092-38d13896fe0c","order_by":2,"name":"Yaohuang Jiang","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yaohuang","middleName":"","lastName":"Jiang","suffix":""},{"id":298064913,"identity":"ed07266c-621b-4f6a-a26f-4c0cc7651559","order_by":3,"name":"Jiehua Qiu","email":"","orcid":"","institution":"China National Rice Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Jiehua","middleName":"","lastName":"Qiu","suffix":""},{"id":298064915,"identity":"f39fdbc4-594c-42f4-8650-c8995c5707ec","order_by":4,"name":"Fei Chen","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Fei","middleName":"","lastName":"Chen","suffix":""},{"id":298064917,"identity":"02c8e1fe-875d-4e9b-9896-3fd130e1e939","order_by":5,"name":"Yanli Zhang","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yanli","middleName":"","lastName":"Zhang","suffix":""},{"id":298064919,"identity":"4e33e32c-8bb5-424e-9170-417a7f068522","order_by":6,"name":"Yu Qiao","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Qiao","suffix":""},{"id":298064921,"identity":"2193c9d1-190d-4826-b1c2-0e905a3c27b3","order_by":7,"name":"Mingyue Tang","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Mingyue","middleName":"","lastName":"Tang","suffix":""},{"id":298064923,"identity":"639c7027-cea6-40b7-bbfc-7fdbf04b0288","order_by":8,"name":"Yingying Ma","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yingying","middleName":"","lastName":"Ma","suffix":""},{"id":298064925,"identity":"5eda2ab3-e2c4-497a-b3f0-48a635532777","order_by":9,"name":"Qian Qian","email":"","orcid":"","institution":"China National Rice Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Qian","suffix":""},{"id":298064927,"identity":"3a491b72-8371-4341-a857-b18fa22c3102","order_by":10,"name":"Limin Wu","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Limin","middleName":"","lastName":"Wu","suffix":""},{"id":298064929,"identity":"46eb888a-bc78-42e9-bf3f-ba75dd595e79","order_by":11,"name":"Yancun Yu","email":"","orcid":"","institution":"Hangzhou Normal University","correspondingAuthor":false,"prefix":"","firstName":"Yancun","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2024-04-26 02:27:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4326724/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4326724/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12284-024-00731-x","type":"published","date":"2024-08-13T15:57:20+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55930971,"identity":"0d0ee20a-5460-4fc3-bcda-0d666ac5b145","added_by":"auto","created_at":"2024-05-06 12:38:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":352585,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePhenotypic comparison of WT and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003espl50\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant plants.\u003c/strong\u003e (A) Overall morphology of WT\u003cstrong\u003e \u003c/strong\u003eand \u003cem\u003espl50\u003c/em\u003emutant at 50 days post-germination. Scale bar = 10 cm. (B) Comparative leaf morphology of WT and \u003cem\u003espl50\u003c/em\u003e at 50-day-old seedlings. (C) Morphological differences between WT\u003cstrong\u003e \u003c/strong\u003eand \u003cem\u003espl50\u003c/em\u003e at the tillering stage. Scale bar = 10 cm. (D) Leaf morphology showcasing WT\u003cstrong\u003e \u003c/strong\u003eand \u003cem\u003espl50\u003c/em\u003e at the tillering stage. (E) Comparison of WT\u003cstrong\u003e \u003c/strong\u003eand \u003cem\u003espl50\u003c/em\u003e plant phenotypes at the maturity stage. Scale bar = 10 cm. (F) Leaf morphology contrast between WT\u003cstrong\u003e \u003c/strong\u003eand \u003cem\u003espl50\u003c/em\u003e plants at the mature stage.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4326724/v1/2ca146b7f2c52f9a18dc12c3.png"},{"id":55931596,"identity":"630ac3ad-08d6-4741-a535-ee497c76b827","added_by":"auto","created_at":"2024-05-06 12:46:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":214629,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular analysis of leaf senescence and ROS-associated parameters in WT and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003espl50\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant lines.\u003c/strong\u003e (A) DAB staining. (B) Trypan blue staining. (C) Relative gene expression levels of senescence-associated markers. (D-F) Quantification of MDA, H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, and O\u003csup\u003e2-\u003c/sup\u003e levels in WT and \u003cem\u003espl50\u003c/em\u003e leaves. (G-I) Enzymatic activity assays for SOD, POD, and CAT in leaves at the heading stage. Data are presented as mean values ± standard deviation (SD) for n=3 biological replicates. Statistical significance was assessed using Student's t-test (*P\u0026lt;0.05, **P\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4326724/v1/3752476f8db48fac5bc300c0.png"},{"id":55930979,"identity":"e3fea0bd-67df-45d5-9214-6779fa1e740a","added_by":"auto","created_at":"2024-05-06 12:38:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":173860,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTUNEL assay for DNA fragmentation in WT and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003espl50\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutants\u003c/strong\u003e. (A-B) TUNEL staining depicting DNA fragmentation. (C-D) DAPI staining indicating total DNA. (E-F) Merged images of TUNEL and DAPI staining. Scale bar = 200 µm.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4326724/v1/9f420a369fdf975de373e38c.png"},{"id":55930981,"identity":"f9222974-ff11-4d11-accf-f68a5231f25a","added_by":"auto","created_at":"2024-05-06 12:38:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":259240,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDisease resistance assessment and defense gene expression analysis in WT and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003espl50\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant lines\u003c/strong\u003e. (A) Pathogenicity assays conducted by spray inoculation with \u003cem\u003eM. oryzae \u003c/em\u003eisolate RO1-1. (B) Quantification of relative lesion area caused by \u003cem\u003eM.\u003c/em\u003e \u003cem\u003eoryzae\u003c/em\u003e in WT and \u003cem\u003espl50\u003c/em\u003e lines. (C) Estimation of relative \u003cem\u003eM\u003c/em\u003e. \u003cem\u003eoryzae\u003c/em\u003e biomass within lesions of WT and \u003cem\u003espl50\u003c/em\u003e. (D) Expression levels of defense signaling-related genes. Values are mean ± SD (n=3). Significant differences determined by Student's t-test (*P\u0026lt;0.05, **P\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4326724/v1/c331c2c0741e3808c30af428.png"},{"id":55930978,"identity":"d04c8480-5686-48ad-bd9f-cccf9c62c182","added_by":"auto","created_at":"2024-05-06 12:38:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":196288,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalysis of chlorophyll content and expression of chloroplast-associated proteins in WT and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003espl50\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mutant lines\u003c/strong\u003e. (A) Comparative chlorophyll content in WT and \u003cem\u003espl50\u003c/em\u003e leaves at the heading stage. (B) Relative expression levels of chloroplast-associated proteins in WT and \u003cem\u003espl50\u003c/em\u003e at the heading stage. (C) Photosynthetic parameter measurements in WT and \u003cem\u003espl50\u003c/em\u003e leaves: A, Net photosynthesis; E, Transpiration rate; gs, Stomatal conductance; Ci, Intercellular CO\u003csub\u003e2\u003c/sub\u003e concentration. Data are presented as means ± SD (n=6), with statistical significance evaluated via Student's t-test (*P\u0026lt;0.05, **P\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4326724/v1/bc1bdb14f535ebefbc374d94.png"},{"id":55930977,"identity":"162a6365-a10b-474c-8452-779360956c09","added_by":"auto","created_at":"2024-05-06 12:38:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":343694,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMap-based cloning of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSPL50\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (A) \u003cem\u003eSPL50\u003c/em\u003e preliminarily mapped between markers B10-1 and B10-8 on chromosome 10. (B-C) Fine mapping of \u003cem\u003eSPL50\u003c/em\u003e. The \u003cem\u003eSPL50\u003c/em\u003elocus was mapped to a 122-kb region between markers P7 and P8. (D) Identification of sixteen candidate ORFs within the122-kb genomic interval. (E) Gene structure of \u003cem\u003eLOC_Os10g05730\u003c/em\u003e. (F) Comparative sequence analysis showing a T-to-A point mutation in \u003cem\u003espl50\u003c/em\u003e relative to the wild type. (G) Amino acid substitution resulting from the mutation, replacing leucine with histidine in the SPL50 protein. (H) Rescue of the \u003cem\u003espl50\u003c/em\u003emutant phenotype through functional complementation.\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4326724/v1/fab6b2f19390035c4df2fafd.png"},{"id":55931599,"identity":"fa1157bc-a948-436f-8bca-c361d375647a","added_by":"auto","created_at":"2024-05-06 12:46:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":247513,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eResistance reactions to \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eMaganaporthe oryzae\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e isolates tested. (\u003c/strong\u003eA) Disease symptoms on leaves of WT, \u003cem\u003espl50\u003c/em\u003e, and pGSPL50 after inoculation with M. oryzae. (B-C) Quantification of \u003cem\u003eM. oryzae\u003c/em\u003e-infected leaf area and fungal biomass in WT, \u003cem\u003espl50\u003c/em\u003e, and pGSPL50 lines. (D-E) Expression analysis of defense-related markers via quantitative reverse transcription PCR (qRT-PCR) in WT, \u003cem\u003espl50\u003c/em\u003e, and pGSPL50. Statistical significance was determined using Student's t-test (*P\u0026lt;0.05, **P\u0026lt;0.01).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4326724/v1/3c4db4fa04ce4ad375488974.png"},{"id":55930984,"identity":"056fa3d9-4aa6-4145-b20c-8881f93832ad","added_by":"auto","created_at":"2024-05-06 12:38:50","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":116337,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression patterns of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eSPL50\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e.\u003c/strong\u003e (A) \u003cem\u003eSPL50\u003c/em\u003e transcript levels in various tissues of WT at booting stage, measured by qRT-PCR. Standard deviations are indicated (n=3). (B-G) Localization of \u003cem\u003eSPL50\u003c/em\u003e promoter-driven GUS reporter gene activity in different tissues: roots (B), stem (C), leaf (D-E), sheath (F), and young panicles (G). Scale bar = 100 µm.\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4326724/v1/6fdba0158cf0ebda4c0de9d5.png"},{"id":55930973,"identity":"00ed4001-c00c-4252-af57-edb0d9612c31","added_by":"auto","created_at":"2024-05-06 12:38:49","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":403742,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSubcellular localization of SPL50\u003c/strong\u003e (A-C) Localization of SPL50 protein in \u003cem\u003eNicotiana benthamiana \u003c/em\u003eleaf cells via GFP fusion protein assay. Scale bar = 50 µm. (D-F) Rice protoplast transient assay showing SPL50 subcellular distribution, with SPL50 tagged with GFP under the control of the 35S promoter. RFP-SDS2 serves as a cytomembrane marker. Scale bar = 20 µm.\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4326724/v1/edc736f743de30e7257efdee.png"},{"id":63071442,"identity":"7648b37e-07da-437a-a5cb-372edf0da298","added_by":"auto","created_at":"2024-08-22 20:07:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3011572,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4326724/v1/fb19f62d-04ab-463f-8f41-1fa46cfa5a74.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"SPL50 negatively regulates cell death and disease resistance in rice","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePlant lesion mimics (LMMs) are a remarkable phenomenon in plant biology that reveal how plants can exhibit symptoms even when they are not experiencing disease or environmental stress (Zhu et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These necrotic spots, varying in shapes and sizes, emerge spontaneously across various plant parts, including the leaves and leaf sheaths. The fact that this phenomenon develops independently of biotic (pathogen-related) or abiotic (environmental stress-related) factors raises the possibility that an underlying genetic or epigenetic mechanism is at work. Numerous plant species exhibit lesion mimic mutants, underscoring a widespread genetic foundation for this phenomenon. Examples include barley (Wolter et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1993\u003c/span\u003e), rice (Takahashi et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1999\u003c/span\u003e), Arabidopsis (Lorrain et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), maize (Mu et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), and wheat (Yu et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang et al., 2023), among others. These mutant`s lesions bear a striking resemblance to those seen in plants after a hypersensitive response (HR), a well-documented form of programmed cell death (PCD) (Cai et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The HR plays a crucial role in controlling the spread of diseases by rapidly eliminating contaminated cells and enclosing the afflicted area to stop the germs from moving further. A key component of the plant's innate immune system, this fast cell death mechanism allows it to successfully fend off pathogen attacks without the involvement of adaptive immunity system, which are present in more complex species.\u003c/p\u003e \u003cp\u003eTo date, an array of LMM genes has been elucidated, encoding proteins that span a diverse range of functions. These include the MEDIATOR SUBUNIT 16 (Zhang et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), the deubiquitinase OsLMP1 (Zou et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Sun et al., 2022), receptor-like protein kinase SPL36 (Rao et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), translation factor OsWRKY19 (Du et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), ATP-citrate lyase SPL30 (Ruan et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), eukaryotic translation elongation factor 1 alpha (eEF1A)-like protein SPL33 (Wang et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), splicing factor 3b subunit SPL5 (Chen et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), and heat shock transcription factor SPL7 (Yamanouchi et al., 2003). Each of these genes plays a distinct and pivotal role in the intricate biological phenomena observed in plants, especially in underlining the mechanisms of disease resistance and PCD in rice.\u003c/p\u003e \u003cp\u003eThe discovery of Armadillo (ARM) repeat proteins, initially identified in \u003cem\u003eDrosophila melanogaster\u003c/em\u003e, signified a noteworthy progression in our comprehension of protein structure and function. These proteins are characterized by a highly conserved structural domain consisting of triplets of α-helices, each arranged in a repeating 42-amino acid pattern (Peifer et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). This unique structural design contributes to their adaptability and allows ARM repeat proteins to assume various conformations, adopting to different functional roles as required. The adaptability of ARM repeat proteins is a crucial aspect of their ability to interact with a wide range of protein partners. This flexibility facilitates their involvement in numerous essential cellular processes, underscoring their importance in maintaining cellular homeostasis and responding to internal and external signals. One critical role of ARM repeat proteins is in nucleo-cytoplasmic transport, which is fundamental to the proper functioning and regulation of cells (Wang et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This process ensures that substances necessary for cellular function are correctly shuttled between the nucleus and the cytoplasm, allowing for the appropriate expression and regulation of genes. Additionally, ARM repeat proteins are vital in mediating cellular responses to environmental cues through signal transduction pathways (Kulich et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). These pathways are essential for the cell's ability to detect and respond to changes in its environment, ensuring cellular adaptation and survival under various conditions. protein trafficking is another crucial process involving ARM repeat proteins (Yoshida et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This mechanism is responsible for the correct transport and localization of proteins within the cell, ensuring that proteins delivered to where they are needed mos. Furthermore, ARM repeat proteins play a significant role in the ubiquitination processes, which tags proteins for degradation or functional modification (Wang et al., 2023; Lv et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eARM repeat proteins are pivotal across a vast array of organisms, playing particularly significant roles in plant biology where they regulate development, stress responses, and metabolic pathways. In \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, the ARM domain-containing protein ARK1 interacts directly with RHD3 to modulate the architecture of the endoplasmic reticulum, a key factor in maintaining cellular integrity and function (Sun et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). This interaction underscores how ARM repeat proteins facilitate the organization of cellular structures. In terms of stress adaptation, ARM repeat proteins like OsPUB2 and its homolog OsPUB3 in rice significantly enhance the plant's tolerance to cold stress, reflecting the functional adaptability of these proteins in response to environmental challenges (Byun et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Additionally, ARM repeat proteins play integral roles in plant defense mechanisms. For example, OsIMα1a and OsIMα1b are crucial in defending rice against blast disease, thereby bolstering crop resilience and food security (Xu et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). The interaction of these proteins with pathogenic challenges illustrates the dynamic role of ARM repeat proteins in mobilizing plant defenses against external threats. Adding to the complexity, proteins like SPL11 and OsPUB15, which possess both ARM and U-box domains, are pivotal in the ubiquitin-mediated regulation of disease resistance mechanisms within rice (Zeng et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Despite extensive research on ARM repeat proteins within the plant kingdom and their proven significance in multiple biological functions, the roles of many members within the ARM family remain largely unexplored. This gap in our knowledge underscores the necessity for ongoing research efforts to elucidate the complex mechanisms through which ARM repeat proteins influence cell death, disease resistance, and other critical processes. Unraveling these mechanisms is essential not only to advance our fundamental understanding of plant biology but also to exploit this knowledge in developing crops with enhanced resistance to stresses and diseases. Such research is crucial for promoting sustainable agriculture and ensuring food security amidst escalating global challenges.\u003c/p\u003e \u003cp\u003eTo investigate the molecular mechanisms governing cell death and disease resistance in rice, we successfully isolated a novel LMM, named \u003cem\u003espotted leaf 50\u003c/em\u003e (\u003cem\u003espl50\u003c/em\u003e), from ethyl methane sulfonate (EMS)-mutagenized \u003cem\u003eOryza sativa japonica\u003c/em\u003e cv. Wuyunjing 7 (WYJ). The \u003cem\u003espl50\u003c/em\u003e mutant displayed distinct spotted leaves beginning at the tillering stage and continuing through the ripening phase, indicative of internal disruptions potentially linked to innate immunity mechanisms. To identify the genetic basis of the \u003cem\u003espl50\u003c/em\u003e phenotype, we employed a map-based cloning strategy. This approach led us to discover that the mutation responsible for the \u003cem\u003espl50\u003c/em\u003e characteristics occurred in a gene encoding an ARM repeat protein. ARM repeat proteins are known for their role in various cellular processes, including developmental regulation and stress response. The mutation in the \u003cem\u003eSPL50\u003c/em\u003e gene led to a notable accumulation of reactive oxygen species (ROS) in the mutant plants. Interestingly, despite the potential for damage, the increased ROS levels in \u003cem\u003espl50\u003c/em\u003e mutants were also associated with enhanced resistance to blast disease. This observation suggests that \u003cem\u003eSPL50\u003c/em\u003e plays a dual role in maintaining ROS homeostasis and activating defense mechanisms against pathogens. This finding adds a new layer to our understanding of SPL50's functions, positioning it as a crucial player in the plant\u0026rsquo;s innate immune response..\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eIdentification of the\u003c/b\u003e \u003cb\u003espl50\u003c/b\u003e \u003cb\u003emutant\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe discovery of the \u003cem\u003espl50\u003c/em\u003e mutant resulted from a meticulous screening process subsequent to the application of EMS (ethyl methane sulfonate) mutagenesis on the \u003cem\u003ejaponica\u003c/em\u003e rice cultivar, Wuyunjing 7 (WYJ). At the seedling stage, the \u003cem\u003espl50\u003c/em\u003e mutant under field conditions showed no noticeable differences from the wild type, including the absence of visible lesions on its leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). As the plants matured from the tillering to the ripening stages, however, pronounced phenotypic differences began to emerge. Both aging and newly formed leaves of the \u003cem\u003espl50\u003c/em\u003e mutants developed reddish-brown lesions, contrasting sharply with the vibrant green foliage observed in wild type plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC-F). This shift in leaf coloration and the appearance of lesions are indicative of significant internal changes within the plant, potentially signaling a hypersensitive response or a self-initiated activation of cell death mechanisms, occuring in the absence of pathogen interaction. Such phenotypic changes suggest that the \u003cem\u003espl50\u003c/em\u003e mutation may have profound effects on the plant\u0026rsquo;s vitality and stress response pathways. Moreover, the \u003cem\u003espl50\u003c/em\u003e mutants exhibited considerable reductions in several agronomic traits compared to their wild type counterparts, such as plant height, grain size, 1000-grain weight, the number of productive panicles, the number of grains per panicle, primary and secondary panicle branches, grain yield per plant, total grain number per plant, and seed setting rate (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAlterations in reactive oxygen species (ROS) metabolism in the\u003c/b\u003e \u003cb\u003espl50\u003c/b\u003e \u003cb\u003emutant\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe appearance of lesion-mimic spots on the leaves of \u003cem\u003espl50\u003c/em\u003e mutants serves as a visible indicator of the activation of cellular death pathways, accompanied by a notable accumulation of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) within the cells. To confirm this phenomenon, we conducted a series of experiments utilizing 3,3'-diaminobenzidine (DAB) staining on both wild type and \u003cem\u003espl50\u003c/em\u003e mutant leaves over an 8-hour period. The results revealed pronounced brown pigmentation exclusively in \u003cem\u003espl50\u003c/em\u003e leaves, indicative of a significant buildup of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, contrasting sharply with the unaltered appearance of wild type leaves (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Furthermore, Trypan blue staining highlighted extensive cellular damage within \u003cem\u003espl50\u003c/em\u003e leaves, diverging markedly from the unaffected wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Quantitative analyses further supported these observations, demonstrating elevated levels of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and superoxide anion (O\u003csup\u003e2\u0026minus;\u003c/sup\u003e) within the \u003cem\u003espl50\u003c/em\u003e mutant, along with increased production of malondialdehyde (MDA), a marker signifying both cellular demise and lipid peroxidation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-F). Enzymatic analyses revealed a decrement in catalase (CAT) activity within \u003cem\u003espl50\u003c/em\u003e, whereas peroxidase (POD) and superoxide dismutase (SOD) activities experienced significant elevation, underscoring an intensified oxidative stress response in the mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-I). Concomitantly, transcriptomic analyses unveiled a significant upregulation of five senescence-associated genes, namely \u003cem\u003eWRKY23\u003c/em\u003e, \u003cem\u003eSGR\u003c/em\u003e, \u003cem\u003eOsI85\u003c/em\u003e, \u003cem\u003eOsh69\u003c/em\u003e, and \u003cem\u003eRCCR1\u003c/em\u003e, in the \u003cem\u003espl50\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). This finding affirms the genetic response to oxidative stress and cellular senescence in \u003cem\u003espl50\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further investigate the cellular death mechanisms in \u003cem\u003espl50\u003c/em\u003e, we examined chromatin condensation within mature leaf cells. the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay can be used to identify the endonucleolytic fragmentation of nuclear DNA, which is the mechanism that is unmistakably associated with programmed cell death (PCD). This analysis unveiled a distinct disparity between the \u003cem\u003espl50\u003c/em\u003e mutants and their wild type counterparts. Specifically, the \u003cem\u003espl50\u003c/em\u003e mutants have a high frequency of TUNEL-positive nuclei, whereas the wild type has a very low frequency of these nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eEnhanced innate immune responses in the\u003c/b\u003e \u003cb\u003espl50\u003c/b\u003e \u003cb\u003emutant\u003c/b\u003e\u003c/p\u003e \u003cp\u003e \u003cem\u003eMagnaporthe oryzae\u003c/em\u003e, the fungus that causes rice blast disease, has caused a drastic decline in cereal yields over 30%, posing a significantly threat to global food security (Zhang et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). In light of this pressing issue, our study sought to assess on evaluating the resistance of the \u003cem\u003espl50\u003c/em\u003e mutant against this formidable pathogen. After two weeks, specimens of both wild type and \u003cem\u003espl50\u003c/em\u003e mutant plants, were inoculated with the \u003cem\u003eM. oryzae\u003c/em\u003e strain R01-1. A week after inoculation, careful monitoring of disease symptoms was undertaken. Significantly, the \u003cem\u003espl50\u003c/em\u003e mutant leaves showed noticeably less lesions than the wild type leaves, which displayed extensive necrotic lesions (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-B). To further substantiate the extent of fungal infiltration and proliferation within the leaf tissues, DNA was meticulously extracted from matched samples of both wild type and \u003cem\u003espl50\u003c/em\u003e mutant leaves. The subsequent quantification of \u003cem\u003eM. oryzae\u003c/em\u003e biomass using quantitative real-time PCR (qRT-PCR) unveiled a significantly diminished fungal biomass in the \u003cem\u003espl50\u003c/em\u003e mutant compared to the wild type (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAdditionally, a comprehensive quantitative analysis of the expression levels of key defense genes, including \u003cem\u003ePR1a\u003c/em\u003e, \u003cem\u003ePR1b\u003c/em\u003e, \u003cem\u003ePR10\u003c/em\u003e, \u003cem\u003ePBZ1\u003c/em\u003e, and \u003cem\u003eSL\u003c/em\u003e, was conducted via qRT-PCR. The results revealed a substantial upregulation of these genes in the \u003cem\u003espl50\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). This robust induction of these defense genes in the \u003cem\u003espl50\u003c/em\u003e mutant provides compelling evidence for its enhanced resistance to the rice blast disease. Our findings underscore the potential of the \u003cem\u003espl50\u003c/em\u003e mutation in bolstering the plant's defense mechanisms against \u003cem\u003eM. oryzae\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eImpact of\u003c/b\u003e \u003cb\u003eSPL50\u003c/b\u003e \u003cb\u003emutation on chloroplast development and photosynthetic efficiency\u003c/b\u003e\u003c/p\u003e \u003cp\u003eLesion mimic phenomena are frequently associated with disruptions to chloroplast ontogeny, aberrant gene expression patterns related to chloroplast, and reduced levels of chlorophyll, all of which have a negative impact on photosynthetic efficiency (Cui et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In an effort to determine if the \u003cem\u003espl50\u003c/em\u003e mutant exhibits similar perturbations, extensive investigations were carried out on heading-stage wild type and \u003cem\u003espl50\u003c/em\u003e plants. This involved quantifying the amount of chlorophyll and assessing the expression of protein linked to chloroplasts in their leaves. As compared to the wild type, our findings showed a considerable drop in the levels of chlorophyll a, chlorophyll b, and carotenoids in the \u003cem\u003espl50\u003c/em\u003e mutant indicating a major impairment in chlorophyll biosynthesis and accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Further, the \u003cem\u003espl50\u003c/em\u003e mutation has a significant influence on the integrity and function of chloroplasts, as demonstrated by the increased degradation of several key proteins associated to chloroplast, including Tic110, CP47, RbcL, D1, and PsbO, as revealed by western blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequent examination of the photosynthetic efficiency within the flag leaves of both genotypes at the heading stage unveiled a marked decline in the net photosynthetic rate, stomatal conductance, and transpiration rate in the \u003cem\u003espl50\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC). This reduction was accompanied by an elevated intracellular CO\u003csub\u003e2\u003c/sub\u003e concentration, indicating a severe disruption in the photosynthetic machinery. These observations provide further evidence that the \u003cem\u003espl50\u003c/em\u003e mutation adversely affects chloroplast functionality in rice, leading to a pronounced decrease in photosynthetic efficiency.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMap-based cloning of\u003c/b\u003e \u003cb\u003eSPL50\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo conduct a genetic analysis of the \u003cem\u003espl50\u003c/em\u003e mutant, we initiated a cross between the mutant phenotype and the wild type cultivar WYJ. Phenotypic examination of all progeny in the F\u003csub\u003e1\u003c/sub\u003e generation revealed wild type characteristics, indicating the recessive nature of the mutation. In the F\u003csub\u003e2\u003c/sub\u003e population, comprising 441 plants, 338 exhibited growth patterns similar to the wild type, while the remaining displayed the mutant phenotype. The observed segregation ratio of 3:1 (\u003cem\u003eχ\u003c/em\u003e\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e0.05\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;0.477\u0026thinsp;\u0026lt;\u0026thinsp;3.841) substantiates the hypothesis that the \u003cem\u003espl50\u003c/em\u003e mutation is governed by a single recessive allele at a nuclear locus.\u003c/p\u003e \u003cp\u003eFor the purpose of identifying the gene associated with the mutant phenotype, the \u003cem\u003espl50\u003c/em\u003e mutant was crossed with an \u003cem\u003eindica\u003c/em\u003e rice variety 9311. From this cross, 1345 F\u003csub\u003e2\u003c/sub\u003e progeny manifesting the mutant phenotype were selected for further analysis. We employed a total of 183 simple sequence repeats (SSRs) and 41 sequence tagged sites (STSs), which are uniformly dispersed across the 12 chromosomes of rice, for genotyping. Initial mapping efforts positioned the mutant locus between markers B10-1 and B10-2 on chromosome 10, as determined by analyzing the 113 F\u003csub\u003e2\u003c/sub\u003e plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Subsequent fine mapping, utilizing a larger set of 1232 F\u003csub\u003e2\u003c/sub\u003e mutants, refined the locus to a 112-kb interval flanked by markers P7 and P8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB-C). According to data from the Rice Genome Annotation Project (RGAP, \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://rice.plantbiology.msu.edu\u003c/span\u003e\u003cspan address=\"http://rice.plantbiology.msu.edu\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e), this interval contains sixteen predicted open reading frames (ORFs). Sequencing of this specific region in the \u003cem\u003espl50\u003c/em\u003e mutant identified a transversion mutation from T to A within an exon of \u003cem\u003eLOC_Os10g05370\u003c/em\u003e, resulting in a Leu-to-His substitution at the 343th amino acid (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD-G). Thus, \u003cem\u003eLOC_Os10g05370\u003c/em\u003e has been identified as the candidate gene responsible for the observed mutant phenotype.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eConfirmation of\u003c/b\u003e \u003cb\u003eLOC_Os10g05370\u003c/b\u003e \u003cb\u003eresponsible for the mutant phenotype of\u003c/b\u003e \u003cb\u003espl50\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo definitively confirm that \u003cem\u003eSPL50\u003c/em\u003e corresponds to \u003cem\u003eLOC_Os10g05370\u003c/em\u003e, we meticulously inserted a 5.1 kb genomic fragment of \u003cem\u003eLOC_Os10g05370\u003c/em\u003e into the binary vector pCAMBIA1300, followed by its introduction into the \u003cem\u003espl50\u003c/em\u003e mutant via transformation. This process yielded 23 independent T\u003csub\u003e0\u003c/sub\u003e transformants. Detailed phenotypic analysis clearly demonstrated that each transformant effectively mitigated the \u003cem\u003espl50\u003c/em\u003e mutant phenotype, as evidenced by an enhanced plant height and the elimination of lesion-mimic spots (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Subsequent exposure of WT, \u003cem\u003espl50\u003c/em\u003e, and the complemented line p\u003cem\u003eGSPL50-1\u003c/em\u003e to the rice blast pathogen underscored the functional restoration of disease resistance. Notably, there were no significant differences in the extent of lesion formation and pathogen biomass accumulation between p\u003cem\u003eGSPL50-1\u003c/em\u003e and WYJ (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-C). In addition, a pronounced reduction in the expression levels of defense-related genes \u003cem\u003eOsPR1a\u003c/em\u003e and \u003cem\u003eOsPR1b\u003c/em\u003e in p\u003cem\u003eGSPL50-1\u003c/em\u003e compared to \u003cem\u003espl50\u003c/em\u003e was observed, indicating a normalization of the defense response (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD-E). These findings provide compelling evidence supporting the hypothesis that the lesion mimic phenotype observed in the \u003cem\u003espl50\u003c/em\u003e mutant is attributable to a single-base substitution mutation within the \u003cem\u003eLOC_Os10g05370\u003c/em\u003e gene of \u003cem\u003eSPL50\u003c/em\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eConstitutive expression of\u003c/b\u003e \u003cb\u003eSPL50\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThrough qRT-PCR analysis, our investigation revealed the broad expression spectrum of \u003cem\u003eSPL50\u003c/em\u003e gene across diverse plant tissues, such as roots, culms, leaves, leaf sheaths, and panicles. Notably, \u003cem\u003eSPL50\u003c/em\u003e exhibited its highest expression level of in leaf tissue, while its lowest expression was observed within the panicle structures (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). To further elucidate the regulatory elements governing \u003cem\u003eSPL50\u003c/em\u003e expression, we engineered a construct by fusing a 2,052-bp genomic fragment, located upstream of SPL50's start codon, with the β-glucuronidase (GUS) reporter gene. Subsequently, this recombinant DNA was introduced into the genome of wild type plants through transformation. Histochemical analysis of GUS activity in the progeny of these transgenic plants revealed enzymatic activity in roots, culms, leaves, and panicles (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). This pattern of GUS expression closely mirrors the expression profile of \u003cem\u003eSPL50\u003c/em\u003e as determined by qRT-PCR, thus corroborating the constitutive expression paradigm of \u003cem\u003eSPL50\u003c/em\u003e across the examined plant tissues.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSubcellular localization of SPL50\u003c/h2\u003e \u003cp\u003eTo elucidate the subcellular localization of SPL50, our study employed a strategic approach involving the creation of a recombinant expression vector (p35S::SPL50-GFP). This vector was ingeniously designed to fuse the SPL50 coding sequence with the green fluorescent protein (GFP) marker, enabling visual tracking of the protein within cells. Subsequently, this construct was introduced into the cellular environment of rice protoplasts and leaf epidermal cells of tobacco (\u003cem\u003eNicotiana benthamiana\u003c/em\u003e), serving as a model system for plant cell biology studies. Utilizing advanced confocal microscopy techniques, we meticulously observed the distribution of the SPL50-GFP fusion protein within these cells. Our findings compellingly demonstrate that SPL50 predominantly localizes to the cell membrane (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). This localization pattern suggests a potential role for SPL50 in membrane-associated processes or signaling pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe novel identification and characterization of the \u003cem\u003eSPL50\u003c/em\u003e gene within \u003cem\u003eOryza sativa\u003c/em\u003e not only enriches our understanding of plant LMMs but also sheds light on the intricate regulatory networks governing PCD and innate immune responses in plants. \u003cem\u003eSPL50\u003c/em\u003e, encoding an ARM repeat protein, emerges as a pivotal regulator orchestrating PCD and enhancing innate immune responses in rice. The \u003cem\u003espl50\u003c/em\u003e mutant's spontaneous development of necrotic lesions, independent of pathogen attack, underscores a self-activated PCD pathway, akin to an autoimmune response. This phenomenon is reflective of the mutant's altered ROS metabolism, characterized by excessive accumulation of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and O\u003csup\u003e2\u0026minus;\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), hallmark indicators of oxidative stress and cell death (Mittler, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFurthermore, the enhanced resistance of the \u003cem\u003espl50\u003c/em\u003e mutant to rice blast disease (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), underscores \u003cem\u003eSPL50\u003c/em\u003e's significant contribution to the plant's immune defense. The observed upregulation of defense-related genes (e.g., \u003cem\u003ePR1a\u003c/em\u003e, \u003cem\u003ePR1b\u003c/em\u003e, \u003cem\u003ePR10\u003c/em\u003e, \u003cem\u003ePBZ1\u003c/em\u003e, and \u003cem\u003eSL\u003c/em\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) in the \u003cem\u003espl50\u003c/em\u003e mutant suggests that \u003cem\u003eSPL50\u003c/em\u003e influences the transcriptional regulation of key components in rice's defense signaling pathways.\u003c/p\u003e \u003cp\u003eThe mechanisms by which SPL50 regulates PCD and immune response likely involve its ARM repeat domains, facilitating protein-protein interactions crucial for various cellular processes, including signal transduction, gene expression regulation, and multiprotein complex assembly (Yoshida et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Wang et al., 2023; Lv et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Kulich et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Wang et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). In this study, it is plausible that its ARM domains interact with specific proteins involved in ROS metabolism and defense signaling pathways. The accumulation of ROS in the \u003cem\u003espl50\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) may be attributed to \u003cem\u003eSPL50\u003c/em\u003e's potential role in modulating antioxidant enzyme activities, such as CAT, POD, and SOD. SPL50 might influence the expression or stability of these enzymes, thereby affecting ROS detoxification and the balance between ROS production and scavenging. SPL50's contribution to enhanced disease resistance could involve the modulation of defense signaling pathways, possibly through interactions with key signal transducers or transcription factors involved in immune response activation. The upregulation of defense-related genes in the \u003cem\u003espl50\u003c/em\u003e mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD) suggests that SPL50 may influence the transcriptional machinery directly or indirectly, perhaps by affecting the stability or activity of transcription factors, which play a central role in plant immune responses.\u003c/p\u003e \u003cp\u003eMoreover, the SPL50-mediated regulation of chloroplast functionality and photosynthetic efficiency (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e) indicates a broader impact of SPL50 on plant physiology, connecting PCD and immune response to photosynthetic performance. This relationship underscores the interconnectedness of plant defense mechanisms with other physiological processes, highlighting the complexity of plant responses to environmental stresses.\u003c/p\u003e \u003cp\u003eIn summary, the \u003cem\u003eSPL50\u003c/em\u003e gene exemplifies the sophisticated regulatory mechanisms plants employ to mediate PCD and innate immune responses. Through its ARM repeat domains, SPL50 likely interacts with a spectrum of proteins to modulate ROS metabolism and defense signaling pathways, playing a crucial role in rice's ability to counteract pathogenic attacks while managing cellular homeostasis. Future studies should aim to elucidate the specific protein-protein interactions involving SPL50, further clarifying its role in plant PCD and immunity. Understanding these mechanisms not only contributes to our fundamental knowledge of plant biology but also offers potential avenues for enhancing crop resistance to diseases, a critical goal in the face of global food security.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe novel identification of the \u003cem\u003eSPL50\u003c/em\u003e gene in rice enriches our understanding of plant lesion mimic phenomena and unveils its pivotal role in regulating PCD and innate immune responses. Encoding an ARM repeat protein, SPL50 orchestrates PCD and enhances immune responses independently of pathogen attack. Its influence on ROS metabolism and defense signaling pathways underscores its significance in rice's defense mechanisms. The upregulation of defense-related genes in the \u003cem\u003espl50\u003c/em\u003e mutant suggests \u003cem\u003eSPL50\u003c/em\u003e's involvement in transcriptional regulation. Additionally, SPL50's impact on chloroplast functionality and photosynthetic efficiency highlights its broader physiological effects. Through protein-protein interactions mediated by its ARM repeat domains, SPL50 likely modulates ROS metabolism and defense signaling pathways, contributing to both disease resistance and cellular homeostasis. Further research elucidating SPL50's specific interactions and mechanisms promises insights into plant biology and avenues for crop improvement, vital for global food security challenges.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eLMMs: Plant lesion mimics; HR: Hypersensitive response; PCD: Programmed cell death; eEF1A: Eukaryotic translation elongation factor 1 alpha; ARM: Armadillo; CAT: Catalase; POD: Peroxidase; SOD: Superoxide dismutase; DAB: 3,3\u0026apos;-diaminobenzidine MDA: Malondialdehyde; EMS: Ethyl methane sulfonate; ROS: Reactive oxygen species; qRT-PCR: Quantitative real-time PCR; SSRs: Simple sequence repeats; STSs: Sequence tagged sites; GUS: \u0026beta;-glucuronidase; GFP: Green fluorescent protein; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling; RGAP: Rice Genome Annotation Project.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by \u0026ldquo;Ten-thousand Talents Plan\u0026rdquo; of Zhejiang Province (2022R52027 to Y.Y.), the funds from the Natural Science Foundation of Zhejiang (LY23C130001 to B.R.) and the National Natural Science Foundation of China (32370328 to L.W.).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eY. Y, Q. Q, and B. R designed the experiments; B. R, H. W, Y. J, J. Q, F. C, Y. Q, M. T, Y. M, and L. W performed the experiments; Y. Y, Q. Q, B. R and H. W analyzed the data; B. R wrote the manuscript. L. W, Y. Y and Q. Q revised the manuscript. All authors read and approved the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eByun MY, Cui LH, Oh TK, Jung YJ, Lee A, Park KY, Kang BG, Kim WT (2017) Homologous U-box E3 Ubiquitin Ligases OsPUB2 and OsPUB3 Are Involved in the Positive Regulation of Low Temperature Stress Response in Rice (\u003cem\u003eOryza sativa\u003c/em\u003e L.). 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Nat Commun 9 (1):2039.\u003c/li\u003e\n\u003cli\u003eZhu X, Ze M, Chern M, Chen X, Wang J (2020) Deciphering rice lesion mimic mutants to understand molecular network governing plant immunity and growth. Rice Sci 27(4): 278-288.\u003c/li\u003e\n\u003cli\u003eZou T, Li G, Liu M, Liu R, Yang S, Wang K, Lu L, Ye Q, Liu J, Liang J, Deng Q, Wang S, Zhu J, Liang Y, Liu H, Yu X, Sun C, Li P, Li S (2023) A ubiquitin-specific protease functions in regulating cell death and immune responses in rice. Plant Cell Environ 46 (4):1312-1326.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"rice","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rice","sideBox":"Learn more about [Rice](http://thericejournal.springeropen.com)","snPcode":"12284","submissionUrl":"https://submission.nature.com/new-submission/12284/3","title":"Rice","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"SPL50, ARM repeat protein, cell death, disease resistance, rice","lastPublishedDoi":"10.21203/rs.3.rs-4326724/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4326724/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe identification of \u003cem\u003espotted leaf 50\u003c/em\u003e (\u003cem\u003espl50\u003c/em\u003e), a novel lesion mimic mutant (LMM) in rice, provides critical insights into the mechanisms underlying programmed cell death (PCD) and innate immunity in plants. Based on ethyl methane sulfonate (EMS)-induced mutagenesis, the \u003cem\u003espl50\u003c/em\u003e mutant mimics hypersensitive responses in the absence of pathogen by displaying spontaneous necrotic lesions after the tillering phase. SPL50, an ARM repeat protein essential for controlling reactive oxygen species (ROS) metabolism and boosting resistance to blast disease, was identified by map-based cloning techniques. This work also demonstrates the detrimental effects of \u003cem\u003espl50\u003c/em\u003e on photosynthetic efficiency and chloroplast development. The crucial significance of SPL50 in cellular signaling and stress response is shown by its localization to the cell membrane and constitutive expression in various plant tissues. Given increasing concerns about global food security, this research underscores the critical role of SPL50 in modulating PCD and fortifying the immune response, contributing to the development of strategies for enhancing crop disease resistance.\u003c/p\u003e","manuscriptTitle":"SPL50 negatively regulates cell death and disease resistance in rice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-05-06 12:38:44","doi":"10.21203/rs.3.rs-4326724/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-21T10:41:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-31T09:06:47+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-05-22T16:58:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"1849950589901930661510029598611293420","date":"2024-05-21T20:50:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"15629865610448734575329260611127453177","date":"2024-05-13T08:58:41+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-05-13T08:46:45+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-30T07:51:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-30T07:51:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Rice","date":"2024-04-26T02:26:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"rice","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rice","sideBox":"Learn more about [Rice](http://thericejournal.springeropen.com)","snPcode":"12284","submissionUrl":"https://submission.nature.com/new-submission/12284/3","title":"Rice","twitterHandle":"@SpringerOpen","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6c2a9f9b-52fc-489f-a022-adb07ecf0ae7","owner":[],"postedDate":"May 6th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-22T19:39:23+00:00","versionOfRecord":{"articleIdentity":"rs-4326724","link":"https://doi.org/10.1186/s12284-024-00731-x","journal":{"identity":"rice","isVorOnly":false,"title":"Rice"},"publishedOn":"2024-08-13 15:57:20","publishedOnDateReadable":"August 13th, 2024"},"versionCreatedAt":"2024-05-06 12:38:44","video":"","vorDoi":"10.1186/s12284-024-00731-x","vorDoiUrl":"https://doi.org/10.1186/s12284-024-00731-x","workflowStages":[]},"version":"v1","identity":"rs-4326724","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4326724","identity":"rs-4326724","version":["v1"]},"buildId":"_2-kVJe1T_tPrBINL-cwx","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}