水稻抗稻瘟病突变体lmm326的鉴定与调控通路初步分析

doi:10.16819/j.1001-7216.2017.7007 335

摘要/Abstract

摘要： 目的

绝大多数水稻类病斑突变体中免疫系统被激活可以有效提升其对稻瘟病的抗性,为进一步解析类病斑突变体抗病的分子机制,对类病斑突变体lmm326进行了研究。

方法

lmm326是粳稻中花11通过EMS辐射诱变,经多代自交和回交获得的一个类病斑突变体,并将突变体分别与中花11和Dular杂交获得的F2群体用于遗传分析,采用图位克隆的方法对目的基因进行精细定位。

结果

5叶期时,该突变体下部叶片表面开始出现类病斑表型;与野生型相比,突变体叶片光合色素含量、净光合速率、株高、结实率、单株有效分蘖数、千粒重等显著下降;突变体带病斑叶片中死亡细胞数量及过氧化氢的积累量明显多于野生型;突变体相较于野生型对4个已鉴定的稻瘟病生理小种的抗性明显提高。遗传分析表明该性状由一对单隐性核基因控制。采用图位克隆方法将该基因定位在第1染色体长臂端38 kb的区间内,其中包含6个开放阅读框。测序发现基因Os01g0919900第2外显子上对应CDS的第433位碱基由C突变成T,最终导致其编码蛋白的第145个氨基酸由苯丙氨酸(F)变为亮氨酸(L)。qRT-PCR结果显示,与野生型相比,lmm326中参与水杨酸信号通路的防卫基因显著上调表达。

结论

LMM326与OsSSI2互为等位基因,该基因可能参与负调控水杨酸信号转导途径,其突变激活了水稻体内的防卫反应。

关键词: 水稻, 稻瘟病, 防卫反应, 类病斑

Abstract:

【Objective】Rice lesion mimic mutants often have a certain resistance to rice blast. To understand the genetic mechanisms behind its blast resistance, we identified the rice lesion mimic mutant lmm326 and analyzed its regulatory pathway. 【Method】 lmm326 was obtained from an EMS-induced Zhonghua 11 mutant bank, and population derived from lmm326/ Zhonghua 11 and lmm326/Dular were used for genetic and fine mapping. 【Result】At 5-leaf stage, there were brown lesions on lower leaves of lmm326 initially. Compared with the wild type(WT), photosynthetic pigment contents, net photosynthetic rate, plant height, seed setting rate, tiller number, and 1000-grain weight of lmm326 were significantly reduced. Evans blue and DAB staining assay indicated that leaves of lmm326 accumulated more dead cells and H2O2. Additionally, lmm326 displayed higher resistance to 4 races of rice blast compared with WT. According to genetic analysis, the phenotype of mutant was controlled by a single recessive gene. Here, we employed map-based cloning approach to finely map LMM326 through F2 population derived from a cross between lmm326 and Dular, and the gene was narrowed to a 38-kb region on the long arm of chr. 1, which harbored six ORFs. Sequence analysis revealed that a single base substitution at position C433T in Os01g0919900 CDS resulted in a substitution of leucine for phenylalanine at position 145. Real-time PCR showed that expression levels of defense-related genes were significantly higher in lmm326 than in WT.【Conclusion】These results demonstrated that the target gene is allelic to OsSSI2, and LMM326 is likely to participate in salicylic acid-signaling pathway, and its mutation activates the defense response.

Key words: rice, rice blast, defense response, lesion mimic

徐婷婷, 余宁, 张迎信, 毕真真, 吴玮勋, 曹永润, 王备芳, 张越, 轩丹丹, 陈代波, 占小登, 程式华, 曹立勇. 水稻抗稻瘟病突变体lmm326的鉴定与调控通路初步分析[J]. 中国水稻科学, 2017, 31(4): 335-344.

Tingting XU, Ning YU, Yingxin ZHANG, Zhenzhen BI, Weixun WU, Yongrun CAO, Beifang WANG, Yue ZHANG, Dandan XUAN, Daibo CHEN, Xiaodeng ZHAN, Shihua CHENG, Liyong CAO. Identification of Rice Blast Resistance Mutant lmm326 and Preliminary Analysis of Its Regulatory Pathway[J]. Chinese Journal OF Rice Science, 2017, 31(4): 335-344.

图/表 10

参考文献 41

[1]	Heath M C.Hypersensitive response-related death.Plant Mol Biol, 2000, 44: 321-334.
[2]	Wang S H, Lim J H, Kim S S, Cho S H, Yoo S C, Koh H J, Sakuraba Y, Paek N C.Mutation of SPOTTEDLEAF3 (SPL3) impairs abscisic acid-responsive signaling and delays leaf senescence in rice, J Exp Bot, 2015, 66(22): 7045-7059.
[3]	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. Cell, 1997, 89: 25-31.
[4]	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. Cell, 1997, 88: 685-694.
[5]	Buschges R, Hollricher K, Panstruga R, Simons G, Wolter M,Frijters A, van Daelen R, van der Lee T, Diergaarde P, Groenendijk J, Töpsch S, Vos P, Salamini F, Schulze-Lefert P. The barley mol gene: A novel control element of plant pathogen resistance.Cell, 1997, 88: 695-705.
[6]	Brodersen P, Petersen M, Pike H M, Olszak B, Skov S, Odum N, Jørgensen 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. Genes Dev, 2002, 16: 490-502.
[7]	Wu C J, Bordeos A, Madamba M R, Baraoidan M, Ramos M, Wang G L, Leach J E, Leung H.Rice lesion mimic mutants with enhanced resistance to diseases.Mol Genet Genom, 2008, 279: 605-619.
[8]	Dangl J L, Dietrich R A, Richberg M H.Death don’t have no mercy: Cell death programs in plant-microbe interactions.Plant cell, 1996, 8(10):1793.
[9]	Neuffer M G, Calvert O H.Dominant disease lesion mimics in maize.J Hered, 1975, 6(5): 265-270.
[10]	Shirasu K, Chuize-Lefert P.Regulators of cell death in disease resistance. Plant Mol Biol, 2000, 44(3): 371-385.
[11]	Li Z, Zhang Y X, Liu L, Liu Q E, Bi Z Z, Yu N, Cheng S H, Cao L Y.Fine mapping of the lesion mimic and early senescence 1(lmes1) in rice(Oryza sativa). Plant Physiol Biochem, 2014, 80: 300-307.
[12]	Thomma B P, Penninckx I A, Broekaert W F, Cammue B P.The complexity of disease signaling in Arabidopsis. Curr Opin Immunol, 2001, 13(1): 63-68.
[13]	Yoshioka K, Kachroo P, Tsui F, Sharma S B, Shah J, Klessig D F.Environmentally sensitive, SA-dependent defense responses in the cpr22 mutant of Arabidopsis. Plant J, 2001, 26(4): 447-459.
[14]	Weymann K, Hunt M, Uknes S, Neuenschwander U, Lawton K, Steiner H Y, Ryals J.Suppression and restoration of lesion formation in Arabidopsis Isd mutants. Plant Cell, 1995, 7(12): 2013-2022.
[15]	Lu H, Salimian S, Gamelin E, Wang G Y, Fedorowski J, LaCourse W, Greenberg J T. Genetic analysis of acd6-1 reveals complex defense networks and leads to identification of novel defense genes inArabidopsis. Plant J, 2009, 58(3): 401-412.
[16]	Chong J L, Pierrel M A, Atanassova R, Werck-Reichhart D, Fritig B, Saindrenan P.Free and conjugated benzoic acid in tobacco plants and cell cultures. Induced accumulation upon elicitation of defense responses and role as salicylic acid precursors. Plant Physiol, 2001, 125: 318-328.
[17]	Lu H, Rate D N, Song J T, Greenberg J T.ACD6, a novel ankyrin protein, is a regulator and an effector of salicylic acid signaling in the Arabidopsis defense response. Plant Cell, 2003, 15(10): 2408-2420.
[18]	Yu D Q, Chen C H, Chen Z X.Evidence for an important role of WRKY DNA binding proteins in the regulation of NPR1 gene expression. Plant Cell, 2001, 13(7): 1527-1540.
[19]	Despré s C, De Long C, Glaze S, Liu E W, Fobert P R. The Arabidopsis NPR1 NIM1 protein enhances the DNA binding activity of a subgroup of the TGA family of bZIP transcription factors. Plant Cell, 2000, 12: 279-290.
[20]	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. Mol Breeding, 2012, 30(2): 939-949.
[21]	Yamanouchi U, Yano M, Lin H, Ashikari M, Yamada K.A rice spotted leaf gene,SPL7, encodes a heat stress transcription factor protein. Proc Natl Acad Sci, 2002, 99: 7530-7535.
[22]	Zeng L R, Qu S H, Bordeos A, Yang C, Baraoidan M, Yan H, Xie Q, Nahm B H, Leung H, Wang G L.Spotted leaf 11, a negative regulator of plant cell death and defense, encodes a ubox/armadillo repeat protein endowed with E3 ubiquitinligase activity. Plant Cell, 2004, 16: 2795-2808.
[23]	Mori M, Tomita C, Sugimoto K, Hasegawa M, Hayashi N, Dubouzet J G, Ochiai H, Sekimoto H, Hirochika H, Kikuchi S.Isolation and molecular characterization of aSpotted leaf 18 mutant by modified activation-tagging in rice. Plant Mol Biol, 2007, 63: 847-860.
[24]	Qiao Y L, Jiang W Z, Lee J, Park B, Choi M S, Piao R H, Woo M O, Roh J H, Han L Z, Paek N C, Seo H S, Koh H J.SPL28 encodes a clathrin-associated adaptor protein complex 1, medium subunitu1(AP1M1) and early senescence in rice(Oryza sativa). New Phytol, 2010, 185(1): 258-274.
[25]	Fekih R, Tamiru M, Kanzaki H, Abe A, Yoshida K, Kanzaki E, Saitoh H, Takagi H, Natsume S, Undan J R, Undan J, Terauchi R.The rice(Oryza sativa L.) LESION MIMIC RESEMBLING, which encodes an AAA-type ATPase, is implicated in defense response. Mol Genet Genom, 2015, 290: 611-622 .
[26]	Yin Z C, Chen J, Zeng L R, Goh M, Leung H, Khush G S, Wang G L.Characterizing rice lesion mimic mutants and identifying a mutant with broad-spectrum resistance to rice blast and bacterial blight.Mol Plant Microbe Interact, 2000, 13(8): 869-876.
[27]	Wang L J, Pei Z Y, Tian Y C, He C Z.OsLSD1, a rice zinc finger protein, regulates programmed cell death and callus differentiation. Mol Plant Microb Interact, 2005, 18: 375-384.
[28]	Sun C H, Liu L C, Tang J Y, Lin A H, Zhang F T, Fang J, Zhang G F, Chu C C.RLIN1,encoding a putative coproporphyrinogen III oxidase, is involved in lesion initiation in rice. J Genet Genomics, 2011, 38: 29-37.
[29]	Sakuraba Y, Rahman M L, Cho S H, Kim Y S, Koh H J, Yoo S C, Paek N C.The rice faded green leaf locus encodes protochlorophyllide oxidoreductase B and is essential for chlorophyll synthesis under high light conditions.Plant J, 2013, 74: 122-133.
[30]	Thordal-Christansen H, Zhang Z G, Wei Y D.Subcellular localization of H2O2 in plants: H2O2 accumulation in papillae and hypersensitive response during the barley powdery mildew interaction. Plant J, 1997, 11: 1187-1194.
[31]	Kong X P, Li D Q.Hydrogen peroxide is not involved in HrpN from Erwinia amylovora-induced hypersensitive cell death in maize leaves. Plant Cell Rep, 2011, 30: 1273-1279.
[32]	Wellbuin A R.The spectral determination of chlorophyll a and b, as well as total carotenoids, using various solvents with spectrophotometer of different resolution.J Plant Physiol, 1994, 144: 307-313.
[33]	Rogers S O, Bendich A J.Extraction of DNA from milligram amounts of fresh, herbarium and mummified plant tissues. Plant Mol Biol, 1985, 5: 69-76.
[34]	陈析丰, 金杨, 马伯军. 水稻类病变突变体及抗病性的研究进展. 植物病理学报, 2011, 41(1): 1-9.
	Chen X F, Jin Y, Ma B J.Progress on the studies of rice lesion mimics and their resistant mechanism to the pathogens.Acta Phytopathol Sin, 2011, 41(1): 1-9. (in Chinese with English abstract)
[35]	Jiao B B, Wang J J, Zhu X D, Zeng L J, Li Q, He Z H.A novel protein RLS1 with NB-ARM domains is involved in chloroplast degradation during leaf senescence in rice.Mol Plant, 2012, 5(1): 205-217.
[36]	Takahashi A, Agrawal G K, Yamazaki M, Onosato K, Miyao A, Kawasaki T, Shimamoto K, Hirochika H.RicePti1a negatively regulates RAR1-dependent defense responses. Plant Cell, 2007, 19: 2940-2951.
[37]	Feng B H, Yang Y, Shi Y F, Shen H C, Wang H M, Huang Q N, Xu X, Lü X G, Wu J L.Characterization and genetic analysis of a novel rice spotted-leaf mutantHM47 with broad-spectrum resistance to Xanthomnas oryzae pv. oryzae. J Integr Plant Biol, 2013, 55(5): 473-483.
[38]	Liu X Q, Li F, Tang J Y, Wang W H, Zhang F X, Wang G D, Chu J F, Yan C Y, Wang T Q, Chu C C, Li C Y.Activation of the jasmonic acid pathway by depletion of the hydroxide lyaseOsHPL3 reveals crosstalk between the HPL and AOS branches of the oxylipin pathway in rice. Plos One, 2012, 7: e50089.
[39]	Jiang C J, Shimono M, Maeda S, Inoue H, Mori M, Hasegawa M, Sugano S, Takatsuji H.Suppression of the rice fatty-acid desaturase geneosssi2 enhances resistance to blast and leaf blight diseases in rice. Mol Plant-Microbe Interact, 2009, 22: 820-829.
[40]	Yang W, Dong R, Liu L, Hu Z B, Li J, Wang Y, Ding X H, Chu Z H.A novel mutant allele ofSSI2 confers a better balance between disease resistance and plant growth inhibition on Arabidopsis thaliana. BMC Plant Biol, 2016, 16: 208.
[41]	Song N, Hu Z R, Li Y H, Li C, Peng F X, Yao Y Y, Peng H R, Ni Z F, Xie C J, Sun Q X.Overexpression of a wheat stearoyl-ACP desaturase(SACPD) geneTaSSI2 in Arabidopsis ssi2 mutant compromise its resistance to powdery mildew. Gene, 2013, 524: 220-227.

标记	前引物	后引物
Marker	Forward primer	Reverse primer
RD0115	GTTGTAGATGTGATTGGAGAA	GACTATGTATGGCACTGTTGA
RM5310	TAGACAAAGCAACGGGTTCC	CGGAAGCAGGAGAATCGTAG
RM3362	AAGTTGAAGCAGTCGCCAAC	GAATTGCGTGGGATATGGAC
ZH4 ZH7	TCCACGAACAAAGACGAG GACTCCGAGCCAGCAAAA	ACGGCACATTATCAACAACA GTCTCCGTGCCCTTGTGC
ZH8	ATGGAGTCGCCTTGTTGA	AAATGTGGCTGGCTGATC
ZH11	TTCGTCTCATTAGCAGCAT	CATTTATTCACTTGCCACAT
Qpr10	GTCCGGGCACCATCTACACC	CAAGCTTCGTCTCCGTCGAGT
Qpal1	TTCAACGCCGACACCT	GTAGAGCGGATACGACCTG
Qaos2	AAGCTGCTGCAATACGTGTACTGG	CGACGAGCAACAGCCTTCCG
WRKY45	GCCGACGACCAGCACGATCACC	ACGAGCCGACGCCGCCCTC
PR1a	CGTGTCGGCGTGGGTGT	GGCGAGTAGTTGCAGGTGATG
PR1b Actin	TACGCCAGCCAGAGGAGC CAGGCCGTCCTCTCTCTGTA	GCCGAACCCCAGAAGAGG AAGGATAGCATGGGGGAGAG

标记	前引物	后引物
Marker	Forward primer	Reverse primer
RD0115	GTTGTAGATGTGATTGGAGAA	GACTATGTATGGCACTGTTGA
RM5310	TAGACAAAGCAACGGGTTCC	CGGAAGCAGGAGAATCGTAG
RM3362	AAGTTGAAGCAGTCGCCAAC	GAATTGCGTGGGATATGGAC
ZH4 ZH7	TCCACGAACAAAGACGAG GACTCCGAGCCAGCAAAA	ACGGCACATTATCAACAACA GTCTCCGTGCCCTTGTGC
ZH8	ATGGAGTCGCCTTGTTGA	AAATGTGGCTGGCTGATC
ZH11	TTCGTCTCATTAGCAGCAT	CATTTATTCACTTGCCACAT
Qpr10	GTCCGGGCACCATCTACACC	CAAGCTTCGTCTCCGTCGAGT
Qpal1	TTCAACGCCGACACCT	GTAGAGCGGATACGACCTG
Qaos2	AAGCTGCTGCAATACGTGTACTGG	CGACGAGCAACAGCCTTCCG
WRKY45	GCCGACGACCAGCACGATCACC	ACGAGCCGACGCCGCCCTC
PR1a	CGTGTCGGCGTGGGTGT	GGCGAGTAGTTGCAGGTGATG
PR1b Actin	TACGCCAGCCAGAGGAGC CAGGCCGTCCTCTCTCTGTA	GCCGAACCCCAGAAGAGG AAGGATAGCATGGGGGAGAG

性状 Trait	野生型 Wild type	突变体 lmm326
株高Plant height/cm	101.6&#x000b1;1.8	63.1&#x000b1;4.0^**
每株有效分蘖数No. of productive panicles per plant	11.7&#x000b1;2.4	3.1&#x000b1;0.4^**
每穗总粒数No. of spikelets per panicle	147.1&#x000b1;17.0	46.9&#x000b1;9.1^**
结实率Seed-setting rate/%	85.5&#x000b1;0.0	77.4&#x000b1;0.0^*
千粒重1000-grain weight/g	23.7&#x000b1;0.9	18.7&#x000b1;0.7^**

性状 Trait	野生型 Wild type	突变体 lmm326
株高Plant height/cm	101.6&#x000b1;1.8	63.1&#x000b1;4.0^**
每株有效分蘖数No. of productive panicles per plant	11.7&#x000b1;2.4	3.1&#x000b1;0.4^**
每穗总粒数No. of spikelets per panicle	147.1&#x000b1;17.0	46.9&#x000b1;9.1^**
结实率Seed-setting rate/%	85.5&#x000b1;0.0	77.4&#x000b1;0.0^*
千粒重1000-grain weight/g	23.7&#x000b1;0.9	18.7&#x000b1;0.7^**

编号 Code	野生型 Wild type	突变体 Mutant
12-26-49-2	4.26&#x000b1;0.08	0.79&#x000b1;0.07^**
12-128-12-1	3.89&#x000b1;0.05	0.82&#x000b1;0.08^**
12-901-48-1	4.45&#x000b1;0.14	0.83&#x000b1;0.08^**
13-600-33-2	4.56&#x000b1;0.08	0.81&#x000b1;0.03^**