SPL36 encodes a receptor-like protein kinase precursor and regulates programmed cell death and defense responses in rice


 The rice (Oryza sativa) spotted leaf 36 (spl36) mutant was identified from an ethyl methanesulfonate–mutagenized Japonica cultivar Yundao population and was previously shown to display a spontaneous cell death phenotype and enhanced resistance to rice bacterial pathogens. Through the analysis of the expression of related genes, we speculate that spl36 is involved in the disease response by up-regulating the expression of defense-related genes. The results of physiological and biochemical experiments indicated that more cell death occurred in the mutant spl36, and the growth and development of the plant were affected. We have isolated SPL36 via a map-based cloning strategy. A single base substitution was detected in spl36, which results in encoding amino acid changes in the SPL36 protein. The predicted SPL36 encodes a receptor-like protein kinase precursor that contains repeated leucine domains and may be involved in stress response of rice. In the salt treatment experiment, we found that the mutant spl36 showed sensitivity to salt. Therefore, SPL36 may negatively regulate salt stress-related responses.

(armadillo) repeat domains, and might undergo ubiquitination and protein-protein interactions in yeast and mammalian systems (Zeng et al., 2002). Finally, the comparison of amino acid sequences showed that the similarity of spl11 with other plant U-box-ARM proteins was mainly limited to the U-box and ARM repeat regions, and a single base substitution was detected in the spl11 mutant gene, which resulted in the premature termination of translation of spl11 proteins. In addition, in vitro ubiquitination assays showed that the spl11 protein had E3 ubiquitin ligase activity which was dependent on the intact U-box domain, indicating that ubiquitination plays a role in plant cell death and defense, which further suggests that spontaneously formed lesion mimics are associated with uncontrolled PCD (Zeng et al., 2004).
Moreover, OsSSI2 encoded fatty acid dehydrogenase (FAD), which also plays a negative role in the rice defense response, resulting in lesion mimics and delayed growth of rice leaves after loss of FAD function (Jiang et al., 2009). Furthermore, mutations in uridine diphosphate-N-acetylglucosamine pyrophosphorylase (UAP1) during glucose metabolism can also lead to the appearance of lesion mimics in rice leaves (Jung et al., 2005). According to the current study, most rice lesion mimic mutants show enhanced resistance to some extent. Among the more than 80 mutants that have been identi ed, 11 mutants such as spl1, spl9, spl10, cdr1, and cdr3 showed enhanced blast resistance (Liu et al., 2004;Yoshimura et al., 1997;Takahashi et al., 1999); 12 mutants such as spl21, spl24, lmes1, hm197, and hm83 showed enhanced bacterial blight resistance (Wu et al., 2008); 19 mutants such as spl14, bl3, and Lmr showed enhanced blast resistance and bacterial blight resistance (Mizobuchi et al., 2002); and mutant lmm1 showed both enhanced blast resistance and sheath blight resistance; among them, mutants spl2, spl3, spl4, spl6, spl7, and ncr1 showed no enhanced resistance, and their resistance was unchanged or even reduced (Kang et al., 2007;Campbell et al., 2005) .  (Walker, 1994;Zhang, 1998). The leucine-rich repeats (LRRs) class of receptor-like protein kinases are a subtype of receptor-like protein kinases. Plants are continuously subjected to biotic and abiotic stresses such as cold, heat, drought, waterlogging, salt, and pests. LRR-type receptor protein kinases are involved in plant stress responses and defense-related processes, and LRR-type receptor protein kinases related to plant disease resistance have drawn great attention. It has been reported that the extracellular domains of proteins encoded by the Cf gene family of tomato leaf mold have LRR structures, and that differences in the amino acid sequences of the LRR motifs of different proteins in the same family are responsible for their speci city of ligand binding (Thomas et al., 1998). The resistance gene FLS2 of Arabidopsis has a similar structure to the extracellular domain of the tomato Cf gene family (Gómez-Gómez et al., 2001). The extracellular LRR structure of rice Xa21, on binding to ligands (avirulent gene products of rice bacterial blight pathogens), can induce intracellular kinase phosphorylation and produce a series of cellular responses that protect the rice from pathogens (Song et al., 1995;Park and Ronald, 2012). These ndings indicate that the LRR structure plays an important role when it binds to pathogens. In addition, Lee et al  found that the LRR type receptor protein kinase OsRLK1 gene of rice could be induced by low temperature and salt stress, and Junga et al (Junga et al., 2004) also found that expression of CALRR1 of pepper could be induced not only by anthrax pathogens but also under abiotic stress conditions such as high salt, abscisic acid (ABA), and wounding. To further explore the signal transduction pathways of LRR-type receptor kinases in response to stress signals, we isolated and characterized a novel rice lesion mimic mutant, spotted leaf 36 (spl36). This mutant shows spots at the tillering stage and enhanced resistance to bacterial blight. We cloned the SPL36 gene by map-based cloning and demonstrated that it encodes a receptor-like protein kinase receptor that is expressed in all tissues and developmental stages and encodes the protein SPL36 located at the plasma membrane. A high degree of cell death, changes in chloroplast structure, and activation of defense-related responses were observed in spl36 mutants. The experimental results indicate that the loss of SPL36 function is responsible for cell death regulation, premature senescence, and defense response activation.

Plant materials and growth conditions
The spotted leaf mutation spl36 was isolated from a methanesulfonate (EMS)-induced mutant library of Yundao rice (wild-type, WT). Hybridization was performed with TN1 as the male parent and mutant, and the F 1 offspring and F 2 population were grown in the rice experimental eld of Zhejiang Normal University, Jinhua City, Zhejiang Province, China, during the summer of 2018 and 2019. The F 2 population of the spl36/ZF802 cross was used for genetic analysis and the F 2 population of spl36/TN1 was used for map-based cloning. The agronomic traits of wild-type and mutant spl36 were also statistically analyzed, including plant height, tiller number, grain number per panicle, seed setting rate, and 1000-grain weight. The results were analyzed using the average of 10 replicates.

Determination of Photosynthetic Parameters and Chlorophyll Content
From 9:30 a.m.to 11:00 a.m. on sunny days, 10 individual plants with relatively uniform growth were harvested. The photosynthetic parameters of the wild-type and mutant were measured by the LI-6400XT portable photosynthesis tester. Three to ve representative ag leaves were treated and measured, and each leaf was measured in triplicate (the mean value was taken as one replicate). During the measurement, red and blue light sources were used, the light intensity was constant at 1200 µmol/m 2 , the temperature was 30 °C, the CO 2 concentration was the concentration in the air, and the humidity was the humidity in the atmosphere. Five wild-type and mutant plants with relatively uniform growth vigor were selected. 0.05 g of the leaves were taken after weighing and then soaked in 25 mL 1:1 ethanol: acetone solution after being cut into pieces; three duplications were set and subject to the darkening reaction for 24 hours, followed by shaking. The absorbance values at 663 nm, 645 nm, and 470 nm were measured with a spectrophotometer, and the photosynthetic pigment content was calculated and statistically analyzed by the t-test.

Histochemical Analysis
The content and concentration of malondialdehyde (MDA), as well as the enzymatic activity and superoxide dismutase of peroxidase (POD) were compliant with the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The contents of MDA and H 2 O 2 as well as the enzymatic activities of SOD and POD were all measured when the phenotype of the tiller stage mutant spl36 had just appeared. Apoptosis was detected by the TUNEL method, FAA xative was prepared before sampling, and xative was added to a 2 mL centrifuge tube (Liang et al., 2018). At the stage when the lesion mutant phenotype was apparent in the mutant spl36 leaves, the leaf tissues showing the phenotype were harvested and the leaves at the corresponding position of the wild-type were harvested, cut into clumps and placed in the 2/3 position of FAA xative for xation. They were then vacuumed until the samples sank to the bottom, sealed with para lm and stored in a refrigerator at 4 °C. The TUNEL apoptosis detection kit (Roche, Cat No.1684817) was used to determine apoptosis in the samples (Inada et al., 1998).
Linkage analysis and mapping of spl36 SSR primers evenly distributed over the 12 chromosomes of rice stored in our laboratory were used to screen the mutants and TN1 for polymorphisms (Supplementary Table 2). Twenty-one F 2 lesion mimic phenotype single plants of spl36/TN1 were used for linkage analysis to preliminarily con rm the chromosomal location of the target gene. A new InDel marker with a relatively good polymorphism was further developed in the mapping interval, and the target gene was precisely mapped with a single plant showing the mutant phenotype in the F 2 segregating population of spl36/TN1. Genomic DNA was extracted using the hexadecyltrimethylammonium bromide (CTAB) method (Wu et al., 1993). The 10 µL PCR system included: DNA template 1 µL, 10 × PCR buffer 1 µL, forward and reverse primers (10 µmol/L) 0.5 µL each, dNTPs 1 µL, rTaq 0.2 µL, with the addition of H 2 O to make up to 10 µL. The PCR ampli cation program was as follows: pre-denaturation at 94 °C for 4 min; denaturation at 94 °C for 30 s, annealing at 55 °C-60 °C for 30 s (temperature varied according to primers), extension at 72 °C for 30 s, 40 cycles; and nally extension at 72 °C for 10 min. PCR products were electrophoresed on a 4% agarose gel, and then photographed and stored in a gel imager and the data were read. Primers used for mapping (Supplementary Table 3).

Vector Construction
For functional complementation of rice spl36 mutants, the complete genomic DNA fragment including the promoter of SPL36 in the wild-type was ampli ed by PCR with primers spl36-CPT-F/36-CPT-R, and then the constructed transformant was generated by inserting the empty binary vector pCAMBIA1300 through Clontech In-Fusion PCR (TaKaRa). The full-length SPL36 open reading frame (ORF) was ampli ed with the primer pair spl36-GFP-F/spl36-GFP-R, and the coding sequence (CDS) of SPL36 was inserted into the binary vector pHQSN containing the 35S promoter (p35S: SPL36) for sublocalization of cells. The SPL36 promoter was constructed into the expression vector pCAMBIA1305.1, and the expression of SPL36 in rice tissues was revealed by using GUS reporter gene. CRISPR Cas9-gRNA vector was constructed by Jiangsu Baig Gene Technology Co., Ltd. All binary constructs were introduced into the corresponding generated wounded tissues by Agrobacterium tumefaciens (EHA105)-mediated method for validation of subsequent assays (see Supplementary Table 4 for required primers). The uorescence of GFP was observed by confocal laser scanning microscopy (Leica TCS SP5, Leica, Germany), and the primers used for vector construction are shown in Supplementary Table 4.

Inoculation Test
The Xanthomonas oryzae pv. oryzae (causal agent for bacterial blight) was inoculated onto the ag leaves of wild-type Yundao and mutant spl36 at the tillering stage. After inoculation, the phenotype of the inoculated leaves was observed at 5 and 10 days and the lesion length were measured and photographed.

Quantitative Real-Time PCR Analysis
Leaf, root, stem, leaf sheath, panicle, and grain samples of wild-type and mutants at each stage were taken and analyzed using RNAprepPure Plant Kit (Cat No. DP441, Tiangen Biotech, Beijing, China) to extract RNA, according to the instructions. The extracted RNA was ampli ed using a ReverTra-Plusreverse transcription kit (Cat No.FSQ-301, Toyobo, Japan) for post-reverse transcription backup. Real-time PCR (qRT-PCR) was used to detect the expression of defense-related genes and the expression of SPL36 in tissues at each stage, with the OsActin gene used as an internal reference (GenBank accession number: NM001058705). Reaction system: 2 µL cDNA template, 10 µL 2 × SYBR qPCR mix, forward and reverse primers 0.8 µL each, with the addition of ddH 2 O to make up to 20 µL. The reaction program was 95 °C for 30 seconds; 95 °C for 5 seconds, 55 °C for 10 seconds; and 72 °C for 5 seconds for 40 cycles. Each reaction was performed in triplicate, and the relative expression of premature senescenceassociated genes was calculated based on 2 − ΔΔ Ct. The real-time PCR instrument was the quantitative uorescence gene ampli cation instrument qTOWER3G (Jena, Germany). Data were analyzed by PSS19.0 software and Excel. The t-test was used for signi cance analysis of differences, and the primers used for qRT-PCR are shown in Supplementary Table 5.

Salt Stress Assay
Plate test: Seeds of full wild-type Yundao and mutant spl36 were selected, washed, spread on 200 mM NaCl MS medium, and cultured at 28 °C under light. In addition, MS medium without NaCl was used as a control. The assay was performed in triplicate, and the germination rate of seeds was observed and counted at each stage. After nine days, root lengths were counted and photographed.
Salt stress assay at seedling stage: The hydroponic wild-type and mutant seedlings were used for about two weeks, and seedlings with approximately the same growth momentum were selected for the assay. Wild-type seedlings and mutant seedlings were transferred to normal nutrient solution and nutrient solution containing 150 mM NaCl for culture, and four days thereafter, salt-stressed seedlings were transferred to normal nutrient solution for recovery culture for three days to observe plant survival rate and determine fresh weight, conductivity, and proline content.

Phenotype of spl36 lesion mimic
Under normal planting conditions in summer, the leaves of spl36 did not change signi cantly from those of the wild type (WT) before the tillering stage. At tillering stage, the lesion mimic appeared in the leaf apex (Fig. 1A). From the tillering stage to the heading stage, these necrotic spots became more severe and gradually spread to the whole leaf (Fig. 1B). To investigate whether spl36 is induced by light like most lesion mimics, mutant spl36 leaves were covered with 2-3 cm aluminum foil at the tillering stage, with the uncovered mutant leaves used as additional controls. After even days, it was observed that no spread of lesion mimics had occurred in the covered area of the covered leaves, while the lesion mimics on the uncovered control leaves (Fig. 1C). This shows that the lesion mimic phenotype arising from mutant spl36 is induced by light. Meanwhile, the main agronomic traits of mutant spl36 such as plant height, grain number per panicle, and 1000-grain weight were signi cantly reduced ( Fig. 1D-I).

SPL36 Gene Regulates Plant Growth and Development
Because of the negative agronomic changes in mutant spl36, and chloroplasts are the main site of photosynthesis. We speculated that the growth and development of the plants were affected after the appearance of the mutant lesion mimic phenotype (Han et al., 2015). We used a transmission electron microscope to observe chloroplast ultrastructure and found that the chloroplasts of mutant spl36 were atrophied and the volume of chloroplasts became smaller, along with disorganized lamellae inside the chloroplasts ( Fig. 2A-D).We speculated that the growth and development of the plants were affected after the appearance of the mutant lesion mimic phenotype. Measurement of the chlorophyll content of wild-type Yundao and mutant spl36 at the tiller peak revealed that both chlorophyll a and chlorophyll b of mutant spl36 were signi cantly reduced compared with the wild-type (Fig. 2E). We further measured the photosynthetic rate of the plants during this period, and the results showed that the net photosynthetic rate of the mutants was signi cantly reduced (Fig. 2F). Therefore, the SPL36 gene regulates plant growth and development through changes in chloroplast structure.

SPL36 Regulates ROS Accumulation and Cell Death in Rice
The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay is designed to detect DNA fragmentation, which is a marker of programmed cell death (Kim et al., 2009). The TUNEL signal in the nuclei of mutant spl36 cells was intense and randomly distributed, whereas only a weak TUNEL signal was detected in the wild type (Fig. 3A-D). In addition, the accumulation of reactive oxygen species (ROS) at high concentrations leads to an oxidative burst, which causes cell damage and even triggers programmed cell death (Kim et al., 2010). The content of H 2 O 2 and the activity of peroxidase (POD) are directly related to the accumulation of ROS. Superoxide dismutase (SOD) plays an important role in scavenging O 2− in plants. Through the detection of H 2 O 2 content, POD activity and SOD activity, it was found that a large amount of H 2 O 2 accumulated in the mutant spl36 (Fig. 3E), while the activities of POD and SOD in the mutant spl36 were signi cantly reduced (Fig. 3G-H). This decrease in enzyme activity would negatively affect the removal of the related peroxide and negative oxygen ions, resulting in the accumulation of ROS. In addition, membrane lipid peroxidation occurs when plant organs age or suffer damage under stress. Malondialdehyde (MDA) is the nal decomposition product of membrane lipid peroxidation, and the content of MDA can re ect the degree of damage in a stressed plant. We found that the MDA content was signi cantly higher in the mutant spl36 than in the wild-type (Fig. 3F). These results indicate that the lesion mimics in spl36 mutants are caused by ROS accumulation and irreversible membrane damage. In addition, loss of SPL36 function triggers the PCD pathway, ultimately leading to the appearance of the spl36 lesion mimic phenotype.

SPL36 Regulates Defense Responses in Rice
It has been reported that most rice lesion mimic mutants have enhanced resistance to pathogens. To investigate whether the resistance of mutant spl36 to rice pathogens was enhanced, we performed an inoculation assay on wild-type Yundao and mutant spl36 at the tillering stage, and used the leaf clipping method to plant the rice bacterial blight strain HM73. Changes in the inoculation site and the length of the lesion mimics were observed at 5 and 10 days after inoculation, respectively. We found that the leaf apex of the wild-type showed obvious necrotic spots at ve days after inoculation, while the mutant did not show obvious disease spots; the length of the wild-type disease spots was signi cantly longer than that of the mutant 10 days after inoculation (Fig. 4A-E). This shows that the resistance to bacterial pathogens is signi cantly enhanced after the emergence of the mutant spl36 disease spots. To further explore the mechanism of enhanced resistance of mutant spl36 to bacterial pathogens, we examined the expression of defense-related genes in wild-type and mutants at the tillering stage by using qRT-PCR, and the results showed that the expression levels of defense genes MAPK12, WRKY53, BIMK2, AOS2, ASP90, LYP6, PR2, PR1a, and PR1b were signi cantly elevated (Fig. 4F). Thus, loss of SPL36-encoded protein function triggers a rice defense response, which leads to enhanced resistance of mutant spl36 to pathogens.

Genetic Analysis and Map-Based Cloning of SPL36 Gene
Mutant spl36 was used as the female parent to be hybridized with ZF802 of the Japonica cultivar TN1. The F 1 plants did not show the phenotype of lesion mimic, and the segregation ratio of normal phenotype and lesion mimic phenotype in the F 2 population was essentially in compliance with the 3: 1 ratio, indicating that the spl36 phenotype is caused by mutations in a single recessive nuclear gene (Supplementary Table 1). A selection of polymorphic markers from 238 insertion and deletion tags mapped 21 F 2 individuals with a lesion mimic phenotype, and we mapped the mutation site to a location between chromosome 12 B12-5 and B12-6 (Fig. 5A). The SPL36 location was further re ned to a location between JHL-3 and JHL-7 by genotyping 148 mutant F2 individuals from the same cross and adding four additional polymorphic tags (Fig. 5B). Using an additional 554 F 2 mutant individuals and four newly developed polymorphic tags, we nally mapped SPL36 to a 60 kb region between markers InDel1 and InDel2 (Fig. 5C). Website inquiry (http://rice.plantbiology.msu.edu/) predicted that the region had 11 open reading frames (ORFs), which included seven expressed proteins and four functional proteins (Fig. 5D). Through sequencing and alignment, we found that the gene LOC_Os12g08180 was mutated (Fig. 5E), and nucleotide C at position 1462 in the coding region of this gene was replaced with T ( Fig. 5F), resulting in the change of the encoded amino acid from arginine to cysteine (Fig. 5G), so LOC_Os12g08180 was used as a candidate gene for SPL36.

Functional complementation of the spl36 mutant with LOC_Os12g08180
To verify whether the single base substitution in LOC_Os12g08180 was associated with the spl36 phenotype, we constructed the vector pGSPL36, which contained genomic DNA fragments including the promoter of the SPL36 gene in wild-type Yundao, and then introduced it into spl36 by Agrobacterium tumefaciens-mediated transformation. The corresponding empty vector pEmV was also transformed as a control. Of the 60 T0 plants which had been transformed, 54 were positive transformants, all of which showed the same normal phenotype as the wild-type (Fig. 6A), while the plants transformed with the control vector showed the same lesion mimic phenotype as the mutant spl36 (Fig. 6B), demonstrating that LOC_Os12g08180 was SPL36, and that the single base substitution in spl36 led to the appearance of the lesion mimic phenotype of the plants.

Expression pattern analysis of SPL36
We used real-time quantitative PCR (qRT-PCR) to analyze the expression of SPL36 in various organs. The results showed that SPL36 was expressed in the organs, with higher expression in leaves, leaf sheaths, and roots and lower expression in stems and panicles. SPL36 expression was signi cantly higher in all organs of mutant spl36 compared to the wild-type Yundao (Fig. 7A). To analyze the spatiotemporal expression pattern of SPL36 more precisely we constructed the vector pSPL36: GUS by fusing the GUS gene with the promoter of SPL36 in the wild-type. We also utilized Agrobacterium tumefaciens-mediated transformation to obtain transgenic plants. We stained various organs of the transgenic positive plants and observed GUS signal maps in various tissues (Fig. 7B-F), which was consistent with the qRT-PCR results. These results suggested that SPL36 was expressed in all organs and at all developmental stages.

Subcellular Localization of SPL36 Protein
To determine the subcellular localization of SPL36, the full-length coding sequence of SPL36 was fused to the N-terminus of green uorescent protein (GFP). When transiently expressed in rice protoplasts, the GFP signal appeared on the plasma membrane (Fig. 8A-D). To verify this observation, we transformed the plasmid containing the SPL36-GFP fusion vector into Nicotiana benthamiana leaves, resulting in the detection of the SPL36-GFP protein on the membrane (Fig. 8E-H). These results show that the SPL36 protein localizes to the membrane.

SPL36 is involved in salt stress-responsive responses in rice
After verifying that the single base substitution of LOC_Os12g08180 was responsible for the lesion mimic phenotype of mutant spl36, we found that this gene encodes a receptor-like protein kinase 2 precursor. To investigate whether SPL36 is involved in stress response-related pathways, we performed a salt stress assay in at dishes and hydroponic seedlings for the wild-type and mutant In the plate assay, in the absence of salt treatment, we found no signi cant difference in the germination rate of mutant spl36 and wild-type over a one-week period. In the case of salt treatment, the germination rate of both mutant and wild-type decreased signi cantly, while the germination rate of the wild-type was also signi cantly lower than that of the mutant. At day 9 of germination we counted the length of the supra-root portion of the salt-treated and control seedlings, and there was no signi cant difference in the length of the supra-root portion between wild-type and mutant in the control group while the length of the supra-root portion of the mutant was signi cantly lower than that of wild-type in the case of salt treatment (Supplementary Fig. 1). In addition, we also treated the wildtype and mutant seedlings hydroponically for four weeks with salt, returning them to normal conditions after three days of treatment. The results showed insigni cant changes in the wild-type after three days of treatment with the phenotype recovering after restoration of normal conditions, while the mutant spl36 showed signi cant leaf bending after the salt treatment while the phenotype did not recover or even died after restoring normal conditions. Our statistical analysis of fresh weight, conductivity as well as nal survival of plants before and after treatment as well as controls revealed that mutant spl36 was more sensitive to salt treatment (Fig. 9). In summary, SPL36 is involved in salt stress-responsive responses in rice.

Discussion
Lesion mimic mutants are extremely important in the study of programmed cell death and defenserelated responses in plant cells. In the present study, we selected a lesion mimic mutant spl36 from a mutant library by mutagenizing wild-type Yundao using EMS. There was no obvious phenotypic difference between this mutant and the wild-type at the seedling stage, and reddish-brown disease spots appeared initially at the leaf apex at the tillering stage and then gradually spread throughout the leaf. We observed the chloroplast ultrastructure of both wild-type and mutant at this stage and measured their photosynthetic rate. The results showed that the appearance of lesion mimics led to signi cant changes in chloroplast structure, as chloroplasts are the main site of plant photosynthesis (Wu et al., 2018), the appearance of lesion mimics affected both the growth and development of the plants. This is also the direct cause of the decline in multiple agronomic traits in mutant plants (Ishikawa et al., 2001). By mapbased cloning we mapped the genes within an interval of 60 Kb, according to the data information of the rice genome database (http://rice.plantbiology.msu.edu/), we found a total of 11 open reading frames (ORFs) within this interval. Seven expressed proteins and four functional proteins were located in this region. The genomic sequences in the mutant and wild-type were ampli ed by PCR. By sequencing alignment and sequencing analysis, we found that nucleotide Cat position 1462 in the coding region of gene LOC_Os12g08180 was replaced with T, resulting in the change of the encoded amino acid from arginine to cysteine. Through a functional complementation assay, we determined that this gene was SPL36. Structural analysis of the protein encoded by this gene showed that SPL36 encodes a receptorlike protein kinase receptor containing multiple leucine-repeat domains (Supplementary Fig. 2). Previous studies have shown that leucine-rich (LRR) type receptor protein kinase (LRK) is closely related to the plant stress and defense responses. The PRK1 gene was isolated from Arabidopsis in 1997 by Hong et al (Hong et al., 1997) and the LRR domain and protein-protein interactions of this gene were related to the interaction, but also to the stress signals that perceived the environment; In 2014, Yang et al (Yang et al., 2014)screened new LRR-RLKs in wild soybeans: GsLRPK, and con rmed that this could improve drought resistance in Arabidopsis; OsGIRL1 showed an up-regulation response when exposed to abiotic stressinduced salt, osmosis, heat, salicylic acid (SA), and abscisic acid (ABA), but a down-regulation response to jasmonic acid (JA) treatment, and the protein localized to the plasma membrane. The biological function of OsGIRL1 was investigated by studying the overexpression of genes during irradiation, salt pressure, osmotic pressure, and thermal stress in Arabidopsis plants (Park et al., 2014). We found, using a germination assay of at dish salt stress treatment and salt treatment assay of hydroponic seedlings, that the mutant spl36 was more sensitive to salt treatment, which may be explained by the fact that a missense mutation of gene LOC_Os12g08180 in the coding region led to the loss of protein function, the speci c mechanism of which remains to be elucidated and which is the main direction of our future study. In previous studies, it has been observed that leucine-rich receptor protein kinases (LRR-LRKs) are mainly associated with abiotic stress responses in plants, while the relationships with PCD and disease resistance have not been reported. We veri ed a higher degree of cell death in the mutant spl36 by the TUNEL assay, and further measured the levels of H 2 O 2 and MDA and the activities of POD and SOD in the wild-type and mutant; the results showed that mutant spl36 accumulated more ROS which led to an oxidative burst and ultimately to PCD. Since lesion mimics of spl36 arise spontaneously, we conclude that SPL36 negatively regulates PCD in rice. In addition, most of the reported lesion mimic mutants showed some disease resistance, and to verify whether SPL36 was involved in the disease resistance response in rice, we inoculated the wild-type and mutant with the bacterial blight pathogen HM73 by the shearing method. and found that the mutant spl36 had signi cant resistance to this pathogen. However, it remains to be determined whether spl36 has broad-spectrum resistance as HM73 is only a bacterial pathogen. At the same time, we analyzed the differences in expression of some defense-related genes in the wild-type and mutants, and the results showed that the expression levels of the defense genes MAPK12, WRKY53, BIMK2, AOS2, LYP6, PR1a, and PR1b were signi cantly elevated. OsWRKY53 is a transcriptional activator that plays an important role in the excitation-induced defense signal transduction pathway in rice (Chujo et al., 2007;Tian et al., 2017). OsAOS2 expression in leaves is signi cantly induced by rice blast and driving OsAOS2 with the PBZ1 promoter activates the expression of other pathogenesis-related genes, thereby increasing the resistance to rice blast (Mei et al., 2006) while OsBIMK2 plays an important role in rice disease resistance responses (Song et al., 2006). LYP6, a protein containing cytolytic enzyme motifs, is a pattern recognition receptor for bacterial peptidoglycan (PGN) and fungal chitin and has a dual role in the recognition of peptidoglycan and chitin in rice innate immunity (Liu et al., 2012). OsPR1a and OsPR1b are pathogenesis-related genes (Agrawal et al., 2000). Therefore, we hypothesize that SPL36 regulates the disease resistance response in rice by up-regulating the expression of defense genes, but the speci c mechanism needs to be further investigated.

Conclusion
We have cloned a novel spotted leaf gene (spl36) in this research,which encodes a receptor-like protein kinase precursor that contains repeated leucine domains and may be involved in stress response of rice. This is the rst report of the involvement of a receptor-like protein kinase in rice disease resistance-related pathways. We have shown that loss of SPL36 function results in enhanced resistance of the mutant to pathogens while enhancing the salt sensitivity of the mutant. Our research is currently conducting an indepth study on whether the mutant spl36 has broad-spectrum resistance to pathogens and the involvement of SPL36 in the mechanism of the salt stress response in rice.

Consent for Publication
Not applicable.

Availability of Data and Materials
All data generated or analyzed during this study are included in this published article and its additional les.

Competing interests
Not applicable.        Salt stress experiment of wild-type and mutant spl36 at seedling stage A: phenotypes before and after 150mM NaCl treatment of wild-type and mutant seedlings; B: fresh weight before and after 150mM NaCl treatment of wild-type and mutant seedlings; C: conductivity before and after 150mM NaCl treatment of wild-type and mutant seedlings; D: Survival rate of wild-type and mutants after 150mM NaCl treatment Genetic and physical maps of the SPL36 gene. A The SPL36 gene was located on chromosome 12 between InDel markers B12-5 and B12-6. B The SPL36 gene was delimited to the JHL-3and JHL-7 interval using 148 F2 mutant individuals; marker names and number of recombinants are shown. C Fine genetic mapping of the SPL36 gene based on 554 mutant F2 individuals. D Eleven putative ORFs were located in an ~60-kb region. E Gene structure LOC_Os12g08180. F Sequence analysis of the C-to-T mutation site in plants of wild type and spl36. G Encoded amino acid from Arginine to Cysteine.

Supplementary Files
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