Phenotypic Identification and Gene Mapping of a Lesion Mimic Mutant lmm7 in Rice

doi:10.16819/j.1001-7216.2022.210711

Abstract

Abstract:

【Objective】The study of lesion mimic mutants (LMMs) is helpful to understand the interaction between growth and defense response in plants.【Method】We herein reported a novel lesion mimic mutant 7 (lmm7) derived from a japonica rice variety FI135 by embryo culture. For deciphering the effect of lmm7 on plant growth, phenotype and agronomic traits were compared between the wild type (WT) and lmm7 plants. The ultrastructure of mesophyll cells and histochemical characterization was done in WT and lmm7 plants. Evaluation of WT and lmm7 was observed for resistance to pathogens was employed to explore the underlying effect of lmm7 on plant defense response. An F₂ population derived from the cross between lmm7 mutant and 9311B was used for genetic analysis and gene mapping.【Result】The seedlings of lmm7 were normal at the seedling stage, but the basal leaves began to exhibit brown spots at the initial tillering stage, which then spread to the whole plant, and the number of mosaic spots increased along with the development of lmm7 plants. Compared with the wild type, agronomic traits including plant height, panicle length, effective panicle number, grain number per panicle, and seed setting rate were significantly decreased in the mutant. However, no significant difference in seed traits and heading date was observed between WT and lmm7 plants. The shading assay indicated that lesion formation in lmm7 was induced by light. The mesophyll cells of lmm7 mutant were seriously chlorotic, and the photosynthetic pigment contents of the mutant were significantly decreased at the heading stage. Histochemical analysis showed that a large amount of hydrogen peroxide (H₂O₂) accumulated at the lesion sites. The observation with a transmission electronic microscope (TEM) demonstrated that the number of chloroplasts in mesophyll cells of lmm7 plants was decreased, the lamellar structure of chloroplasts was seriously damaged, and osmiophilic granules were obviously increased in the mutant. Moreover, the levels of reactive oxygen species (ROS) were increased in the internal and surrounding regions of the lesions. The lmm7 plants displayed higher resistance to rice blast compared to the wild type. Genetic analysis revealed that in lmm7 the mutation was controlled by a single recessive gene. Using a map-based cloning strategy, lmm7 was narrowed down to a 260 kb region between InDel markers 7B35 and 7B43 on the short arm of chromosome 7 in rice. Sequencing analysis revealed that a deletion of T base at 2891 nucleotide position occurred in the DNA sequence of LOC_Os07g0203700, resulting in a frame-shift mutation and a premature stop codon.【Conclusion】The results reveal the target gene is allelic to spl5, and the mutation of lmm7 enhances resistance to rice blast, while it curbs the growth and development of lmm7 plants.

Key words: Oryza sativa L., lesion mimic mutants, phenotypic identification, gene mapping

摘要：

【目的】对水稻类病斑突变体的研究有助于解析其与植物生长和防御反应的关系。【方法】本研究在粳稻品系FI135胚培养过程中获得了1个类病斑突变体lmm7(lesion mimic mutant 7)。通过对其进行系统的表型鉴定、农艺性状考查、超微结构观察、生理学特性分析，阐明LMM7基因对植物生长的调控。通过病原菌抗性鉴定，明确lmm7对植物防御反应的影响。利用9311B与突变体lmm7杂交所得F₂群体对该突变体进行了遗传分析和基因精细定位。【结果】该突变体苗期表型正常，分蘖初期，植株基部叶片从叶尖开始不断出现褐色斑点，并向整株扩散，且斑点数目随植株生长不断增加。与野生型相比，突变体的株高、穗长、有效穗数、每穗粒数、结实率及剑叶长宽都显著降低，但籽粒性状和抽穗期没有显著性差异。遮光处理表明，突变体lmm7的表型受到光照诱导，抽穗期突变体lmm7叶肉细胞严重失绿，光合色素含量显著降低。组织化学分析表明，突变体病斑处的H₂O₂含量显著升高。透射电镜观察结果表明，突变体lmm7叶肉细胞的叶绿体数目减少，叶绿体类囊体片层结构严重受损，细胞器肿胀解体，并出现大量嗜锇小体，同时病斑内部和周围区域积累了大量的ROS。抗性鉴定结果显示突变体lmm7稻瘟病抗性水平显著高于野生型。遗传分析表明lmm7的突变表型受单个隐性基因控制。利用图位克隆的方法，目的基因被定位于水稻第7染色体短臂两InDel标记7B35和7B43之间，区间范围约260 kb。测序结果表明该区间内候选基因LOC_Os07g0203700第2891位碱基T发生了单碱基缺失，导致后续移码突变及翻译提前终止。【结论】lmm7与spl5互为等位基因，其突变抑制了植株的生长，同时增强了对稻瘟病的抗性。

关键词: 水稻, 类病斑突变体, 表型鉴定, 基因定位

SUN Zhiguang, DAI Huimin, CHEN Tingmu, LI Jingfang, CHI Ming, ZHOU Zhenling, LIU Yan, LIU Jinbo, XU Bo, XING Yungao, YANG Bo, LI Jian, LU Baiguan, FANG Zhaowei, WANG Baoxiang, XU Dayong. Phenotypic Identification and Gene Mapping of a Lesion Mimic Mutant lmm7 in Rice[J]. Chinese Journal OF Rice Science, 2022, 36(4): 357-366.

孙志广, 代慧敏, 陈庭木, 李景芳, 迟铭, 周振玲, 刘艳, 刘金波, 徐波, 邢运高, 杨波, 李健, 卢百关, 方兆伟, 王宝祥, 徐大勇. 水稻类病斑突变体lmm7的鉴定与基因定位[J]. 中国水稻科学, 2022, 36(4): 357-366.

Figures/Tables 7

Fig. 1. Phenotypic identification of WT plants and lmm7. A, Plants of wild type (WT) and lmm7 mutant at the tillering stage, red arrows indicate lesion mimic leaves; B, Basal leaves of wild type (WT) and lmm7 at the tillering stage; C, Plants of wild type (WT) and lmm7 at the mature period in field conditions; D, Comparisons of the size of mature seeds from WT and lmm7 plants; E-F: Panicles and internodes of main culms. P, Panicle; I to VI indicate corresponding internodes from top to bottom. Bars =5 cm in A; 2 cm in B; 10 cm in C, E; 10 mm in D.

Table 1. Agronomic traits comparison between WT plants and lmm7.

性状 Trait	野生型Wild type	突变体 lmm7	性状 Trait	野生型Wild type	突变体 lmm7
抽穗期 Heading date /d	95.3±0.6	95.7±0.6	千粒重 1000-grain weight/g	24.6±0.4	23.8±0.5
株高 Plant height /cm	102.1±2.6	80.8±1.8^**	剑叶长 Flag leaf length/cm	24.5±1.4	18.8±1.2^**
穗长 Panicle length /cm	17.4±0.8	13.0±1.2^**	剑叶宽 Flag leaf width/cm	1.8±0.3	1.6±0.2^**
有效穗数 Effective panicle	9.2±1.3	6.5±0.7^*	粒长 Grain length/mm	7.6±0.4	7.5±0.6
每穗粒数 Grains per panicle	142.3±6.5	115.4±7.6^**	粒宽 Grain width /mm	4.4±0.2	4.3±0.1
结实率 Seed setting rate /%	90.6±2.5	82.7±4.3^*	粒厚 Grain thickness /mm	2.1±0.2	2.1±0.2

Fig. 2. Photosynthetic pigments contents of the wild type and the lmm7 mutant at heading stage. A, Photosynthetic pigment contents of flag leaf of the wild type and lmm7 mutant at the heading stage; B, Photosynthetic pigment contents of the second leaf from top; C, Photosynthetic pigment contents of the third leaf from top; *,** significantly different at 0.05 and 0.01 levels, respectively.

Fig. 3. Ultrastructure of mesophyll cells in the wild type (WT) and the lmm7 mutant at mature period. A, Flag leaf of WT at mature period; B-D, Ultrastructure of the blade of WT; E, Flag leaf of lmm7 mutant at mature period; F-G, Ultrastructure of the blade of lmm7 mutant; Chl, Chloroplast; Os, Osmiophilic granule; TM, Thylakoid membranes. Bars: 20 μm (B, F), 6 μm (C, G), 3 μm (D, H).

Fig. 4. Shading treatment and histochemical analysis of the wild type and lmm7 mutant. A, Effects of shading on the wild type(WT) and the lmm7 mutant leaves 7 days after shading; Leaves of WT and the lmm7 mutant stained by trypan blue(B) and DAB(C). Bars =1 cm in A, B and C.

Fig. 5. Resistance evaluation of wild type (WT) and lmm7 mutant to rice black-streaked dwarf virus disease (RBSDVD) and rice blast under artificial inoculation condition. A, Disease incidence of wild type (WT) and lmm7 mutant to rice black-streaked dwarf virus disease (RBSDVD) by the artificial inoculation. B, Disease index of wild type (WT) and lmm7 to rice blast by the artificial inoculation. *P < 0.05 by Student’s t-test.

Fig. 6. Molecular mapping of lmm7 on chromosome 7.

References 49

[1]	王晨, 王备芳, 张迎信, 曹永润, 张越, 江敏, 边康吉, 张小惠, 刘群恩. 水稻类病斑突变体lm8015-2的鉴定与基因的精细定位[J]. 中国水稻科学, 2021, 35(4): 352-358.
	Wang C, Wang B F, Zhang Y X, Cao Y R, Zhang Y, Jiang M, Bian K J, Zhang X H, Liu Q E. Identification and gene mapping of a lesion mimic mutant lm8015-2 in rice[J]. Chinese Journal of Rice Science, 2021, 35(4): 352-358. (in Chinese with English abstract)
[2]	Hu G, Richter T E, Hulbert S H, Pryor T. Disease lesion mimicry caused mutations in the rust resistance gene rp1[J]. Plant Cell, 1996, 8(8): 1367-1376.
[3]	Wu C J, Bordeos A, Madamba M R S, Baraoidan M, Ramos M, Wang G L, Leach J E, Leung H. Rice lesion mimic mutants with enhanced resistance to diseases[J]. Molecular Genetics and Genomics, 2008, 279: 605-619.
[4]	Shirsekar G S, Vega-Sanchez M E, Bordeos A, Baraoidan M, Swisshelm A, Fan J, Park C H, Leung H, Wang G L. Identification and characterization of suppressor mutants of spl11-mediated cell death in rice[J]. Molecular Plant-Microbe Interactions, 2014, 27: 528-536.
[5]	Wang S, Lei C L, Wang J L, Ma J, Tang S, Wang C L, Zhao K J, Tian P, Zhang H, Qi C Y, Cheng Z J, Zhang X, Guo X P, Liu L L, Wu C Y, Wan J M. SPL33, encoding an eEF1A-like protein, negatively regulates cell death and defense responses in rice[J]. Journal of Experimental Botany, 2017, 68: 899-913.
[6]	Hofius D, Tsitsigiannis D I, Jones J D, Mundy J. Inducible cell death in plant immunity[J]. Seminars in Cancer Biology, 2007, 17: 166-187.
[7]	Dietrich R A, Richberg M H, Schmidt R, Dean C, Dangl J L. A novel zinc finger protein is encoded by the Arabidopsis LSD1 gene and functions as a negative regulator of plant cell death[J]. Cell, 1997, 88: 685-694.
[8]	Hoisington D A, Neuffer M G, Walbot V. Disease lesion mimics in maize: I. Effect of genetic background, temperature, developmental age, and wounding on necrotic spot formation with Les1[J]. Developmental Biology, 1982, 93(2): 381-388.
[9]	Yao Q, Zhou R H, Fu T H, Wu W R, Zhu Z D, Li A L, Jia J Z. Characterization and mapping of complementary lesion-mimic genes lm1 and lm2 in common wheat[J]. Theoretical and Applied Genetics, 2009, 119: 1005-1012.
[10]	Spassieva S, Hille J. A lesion mimic phenotype in tomato obtained by isolating and silencing an Lls1, homologue[J]. Plant Science, 2002, 162: 543-549.
[11]	Wolter M, Hollricher K, Salamini F, Schulze-Lefert P. The mlo resistance alleles to powdery mildew infection in barley trigger a developmentally controlled defence mimic phenotype[J]. Molecular and General Genetics, 1993, 239: 122-128.
[12]	张宏根, 王茂宇, 张丽佳, 胡雅, 马佳琦, 张翼帆, 汤述翥, 梁国华, 顾铭洪. 水稻类病斑突变体wy3的鉴定和基因定位[J]. 中国水稻科学, 2016, 30(3): 239-246.
	Zhang H G, Wang M Y, Zhang L J, Hu Y, Ma J Q, Zhang Y F, Tang S Z, Liang G H, Gu M H. Characterization and gene mapping of lesion mimic mutant wy3 in rice[J]. Chinese Journal of Rice Science, 2016, 30(3): 239-246. (in Chinese with English abstract)
[13]	Buschges R, Hollricher K, Panstruga R, Simons G, Wolter M, Frijters A, Daelen R, Lee T, Diergaarde P, Groenendijk J, Topsch S, Vos P, Salamini F, Schulze-Lefert P. The barley mlo gene: A novel control element of plant pathogen resistance[J]. Cell, 1997, 88: 695-705.
[14]	Jambunathan N, Siani J M, Mcnellis T W. A humidity sensitive Arabidopsis copine mutant exhibits precocious cell death and increased disease resistance[J]. Plant Cell, 2001, 13: 2225-2240.
[15]	Lorrain S, Lin B, Auriac M C, Kroj T, Saindrenan P, Nicole M, Balague C, Roby D. Vascular associated death1, a novel GRAM domain-containing protein, is a regulator of cell death and defense responses in vascular tissues[J]. Plant Cell, 2004, 16: 2217-2232.
[16]	Noutoshi Y, Kuromori T, Wada T, Hirayama T, Kamiya A, Imura Y, Yasuda M, Nakashita H, Shirasu K, Shinozaki K. Loss of Necrotic Spotted Lesions 1 associates with cell death and defense responses in Arabidopsis thaliana[J]. Plant Molecular Biology, 2006, 62: 29-42.
[17]	Balague C, Lin B, Alcon C, Flottes G, Malmstrom S, Kohler C, Neuhaus G, Pelletier G, Gaymard F, Roby D. HLM1, an essential signaling component in the hypersensitive response, is a member of the cyclic nucleotide-gated channel ion channel family[J]. Plant Cell, 2003, 15: 365-379.
[18]	Rostoks N, Schmierer D, Mudie S, Drader T, Brueggeman R, Caldwell D G, Waugh R, Kleinhofs A. Barley necrotic locus nec1 encodes the cyclic nucleotide-gated ion channel 4 homologous to the Arabidopsis HLM1[J]. Molecular Genetics and Genomics, 2006, 275: 159-168.
[19]	Mosher S, Moeder W, Nishimura N, Jikumaru Y, Joo S H, Urquhart W, Klessig D F, Kim S K, Nambara E, Yoshioka K. The lesion-mimic mutant cpr22 shows alterations in abscisic acid signaling and abscisic acid insensitivity in a salicylic acid-dependent manner[J]. Plant Physiology, 2010, 152: 1901-1913.
[20]	Wang L, Pei Z, Tian Y, He C. OsLSD1, a rice zinc finger protein, regulates programmed cell death and callus differentiation[J]. Molecular Plant Microbe Interactions, 2005, 18: 375-384.
[21]	Yamanouchi U, Yano M, Lin H, Ashikari M, Yamada K. A rice spotted leaf gene, Spl7, encodes a heat stress transcription factor protein[J]. Proceedings of National Academy of Sciences of the USA, 2002, 99: 7530-7535.
[22]	Zeng L, Qu S, Bordeos A, Yang C, Baraoidan M, Yan H, Xie Q, Nahm B H, Leung H, Wang G. 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[J]. Plant Cell, 2004, 16: 2795-2808.
[23]	Qiao Y, Jiang W, Lee J, Park B, Choi M S, Piao R, Woo M O, Roh J H, Han L, Paek N C, Seo H S, Koh H J. SPL28 encodes a clathrin-associated adaptor protein complex 1, medium subunit micro 1 (AP1M1) and is responsible for spotted leaf and early senescence in rice (Oryza sativa)[J]. New Phytologist, 2010, 185: 258-274.
[24]	Kachroo P, Shanklin J, Shah J, Whittle E J, Klessig D F. A fatty acid desaturase modulates the activation of defense signaling pathways in plants[J]. Proceedings of National Academy of Sciences of the USA, 2001, 98: 9448-9453.
[25]	Brodersen P, Petersen M, Pike H M, Olszak B, Skov S, Odum N, Jorgensen L B, Brown R E, Mundy J. Knockout of Arabidopsis accelerated-cell-death11 encoding a sphingosine transfer protein causes activation of programmed cell death and defense[J]. Genes & Development, 2002, 16: 490-502.
[26]	Hu G, Yalpani N, Briggs S P, Johal G S. A porphyrin pathway impairment is responsible for the phenotype of a dominant disease lesion mimic mutant of maize[J]. Plant Cell, 1998, 10: 1095-1105.
[27]	Ishikawa A, Okamoto H, Iwasaki Y, Asahi T. A deficiency of coproporphyrinogen III oxidase causes lesion formation in Arabidopsis[J]. Plant Journal, 2001, 27: 89-99.
[28]	Gray J, Close P S, Briggs S P, Johal G S. A novel suppressor of cell death in plants encoded by the Lls1 gene of maize[J]. Cell, 1997, 89: 25-31.
[29]	Undan J R, Tamiru M, Abe A, Yoshida K, Kosugi S, Takagi H, Yoshida K, Kanzaki H, Saitoh H, Fekih R. Mutation in OsLMS, a gene encoding a protein with two double-stranded RNA binding motifs causes lesion mimic phenotype and early senescence in rice (Oryza sativa L.)[J]. Genes & Genetic Systems, 2012, 87(3): 169-179.
[30]	Wang Z, Wang Y, Hong X, Hu D, Liu C, Yang J, Li Y, Huang Q, Feng Y, Gong H, Li Y, Fang G, Tang H, Li Y,. Functional inactivation of UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1) induces early leaf senescence and defence responses in rice[J]. Journal of Experimental Botany, 2015, 66: 973-987.
[31]	Lichtenthaler H K. Chlorophylls and carotenoids: Pigments of photosynthetic biomembranes[J]. Methods in Enzymology, 1987, 148: 350-382.
[32]	Fang L K, Li Y F, Gong X P, Sang X C, Ling Y H, Wang X W, Cong Y F, He G H. Genetic analysis and gene mapping of a dominant presenescing leaf gene PSL3 in rice (Oryza sativa L.)[J]. Chinese Science Bulletin, 2010, 55: 2517-2521.
[33]	刘永锋, 陆凡, 陈志谊, 吉建安, 陈毓苓, 高渊. 江苏省水稻新品种(系)对稻瘟病的抗性评价[J]. 中国水稻科学, 2002, 16(1): 96-98.
	Liu Y F, Lu G, Chen Z Y, Ji J A, Chen Y L, Gao Y. Resistance evaluation of new rice varieties (lines) to rice blast in Jiangsu Province[J]. Chinese Journal of Rice Sciences, 2002, 16(1): 96-98.
[34]	International Rice Research Institute. Standard Evaluation System for Rice (SES). Los Baños, Philippines: International Rice Research Institute, 2002: 14-18.
[35]	Zhou T, Du L, Wang L J, Wang Y, Gao C Y, Lan Y, Sun F, Fan Y J, Wang G L, Zhou Y J. Genetic analysis and molecular mapping of QTLs for resistance to rice black-streaked dwarf disease in rice[J]. Scientific Reports, 2015, 5: 10509.
[36]	Zhu X B, Ze M, Chen M S, Chen X W, Wang J. Deciphering rice lesion mimic mutants to understand molecular network governing plant immunity and growth[J]. Rice Science, 2020, 27(4): 278-288.
[37]	奉保华, 杨杨, 施勇烽, 林璐, 陈洁, 黄奇娜, 魏彦林, LEUNG H, 吴建利. 水稻淡褐斑叶突变体lbsl1的遗传分析与基因定位[J]. 中国水稻科学, 2012, 26(3): 297-301.
	Feng B H, Yang Y, Shi Y F, Lin L, Chen J, Huang Q N, Wei Y L, Leung H, Wu J L. Genetic analysis and gene mapping of a light brown spotted leaf mutant in rice[J]. Chinese Journal of Rice Sciences, 2012, 26(3): 297-301. (in Chinese with English abstract)
[38]	王建军, 朱旭东, 王林友, 张利华, 薛庆中, 何祖华. 水稻类病斑突变体的生理与遗传分析[J]. 植物生理与分子生物学学报, 2004, 30(3): 331-338.
	Wang J J, Zhu X D, Wang L Y, Zhang L H, Xue Q Z, He Z H. Physiological and genetic analysis of lesion resembling disease mutants (lrd) of Oryza sativa L[J]. Journal of Plant Physiology and Molecular Biology, 2004, 30(3): 331-338. (in Chinese with English abstract)
[39]	Yoshimura A, Ideta O, Iwata N. Linkage map of phenotype and RFLP markers in rice[J]. Plant Molecular Biology, 1997, 35: 49-60.
[40]	Chern M, Xu Q F, Bart R S, Bai W, Ruan D L, Sze-To W H, Canlas P E, Jain R, Chen X W, Ronald P C. A genetic screen identifies a requirement for cysteine-rich- receptor-like kinases in rice NH1 (OsNPR1)-mediated immunity[J]. PLoS Genetics, 2016, 12(5): e1006049.
[41]	Tang J Y, Zhu X D, Wang Y Q, Liu L C, Xu B, Li F, Fang J, Chu C C. Semi-dominant mutations in the CC-NB-LRR-type R gene, NLS1, lead to constitutive activation of defense responses in rice[J]. Plant Journal, 2011, 66: 996-1007.
[42]	Li Z Q, Ding B, Zhou X P, Wang G L. The rice dynamin related protein OsDRP1E negatively regulates programmed cell death by controlling the release of cytochrome c from Mitochondria[J]. PLoS Pathogens, 2017, 13(1): e1006157.
[43]	Chen X F, Pan J W, Cheng J, Jiang G H, Jin Y, Gu Z M, Qian Q, Zhai W X, Ma B J. Fine genetic mapping and physical delimitation of the lesion mimic gene spotted leaf 5 (spl5) in rice (Oryza sativa L.)[J]. Molecular Breeding, 2009, 24(4): 387.
[44]	Chen X F, Hao L, Pan J W, Zheng X X, Jiang G H, Jin Y, Gu Z M, Qian Q, Zhai W X, Ma B J. SPL5, a cell death and defense related gene, encodes a putative splicing factor 3b subunit 3 (SF3b3) in rice[J]. Molecular Breeding, 2012, 30(2): 939-949.
[45]	Babu R, Jiang C J, Xu X, Kottapalli K R, Takatsuji H, Miyao A, Hirochika H, Kawasaki S. Isolation, fine mapping and expression profiling of a lesion mimic genotype, spl^(NF4050-8) that confers blast resistance in rice[J]. Theoretical and Applied Genetics, 2011, 122: 831-854.
[46]	Li Z, Zhang Y, Liu L, Liu Q, Bi Z, Yu N, Cheng S, Cao L. Fine mapping of the lesion mimic and early senescence 1 (lmes1) in rice (Oryza sativa)[J]. Plant Physiology and Biochemistry, 2014, 80: 300-307.
[47]	夏赛赛, 崔玉, 李凤菲, 谭佳, 谢园华, 桑贤春, 凌英华. 水稻类病斑早衰突变体lmps1的表型鉴定与基因定位[J]. 作物学报, 2019, 45(1): 46-54.
	Xia S S, Cui Y, Li F F, Tan J, Xie Y H, Sang X C, Ling Y H. Phenotypic characterizing and gene mapping of a lesion mimic and premature senescence 1 (lmps1) mutant in rice (Oryza sativa L.)[J]. Acta Agronomica Sinica, 2019, 45(1): 46-54. (in Chinese with English abstract)
[48]	郭丹, 施勇烽, 王惠梅, 张晓波, 宋莉欣, 徐霞, 贺彦, 郭梁, 吴建利. 一个水稻显性斑点叶突变体的鉴定和基因精细定位[J]. 作物学报, 2016, 42(7): 966-975.
	Guo D, Shi Y F, Wang H M, Zhang X B, Song L X, Xu X, Guo L, Wu J L. Characterization and gene fine mapping of a rice dominant spotted-leaf mutant[J]. Acta Agronomica Sinica, 2016, 42(7): 966-975. (in Chinese with English abstract)
[49]	Sun L T, Wang Y H, Liu L L, Wang C M, Gan T, Zhang Z Y, Wang Y L, Wang D, Niu M, Long W H, Li X H, Zheng M, Jiang L, Wan J M. Isolation and characterization of a spotted leaf 32 mutant with early leaf senescence and enhanced defense response in rice[J]. Scientific Reports, 2017, 7: 41846.