大豆根瘤菌HH103 rhcN突变对结瘤能力的影响

doi:10.3864/j.issn.0578-1752.2021.06.003

摘要/Abstract

摘要：

【背景】大豆是重要的经济作物,能够为人类提供大量的植物蛋白和油脂。大豆在生长过程中,能够与根瘤菌形成共生体系进行共生固氮,该形式的共生固氮能够将空气中的氮气还原成氨,为大豆生长发育提供丰富氮源。根瘤菌Ⅲ型分泌系统是根瘤菌中重要的蛋白分泌结构,能够通过分泌Ⅲ型效应因子在共生体系建立过程中发挥重要调控作用。根瘤菌rhcN编码一种ATP转移酶,是根瘤菌Ⅲ型分泌系统的重要组成部分,能够保障根瘤菌Ⅲ型效应因子向宿主细胞的正常分泌。【目的】探索rhcN突变情况下对根瘤菌结瘤能力的影响,提高大豆-根瘤菌共生体系的固氮效率,为大豆农业生产提供必要的参考和支持。【方法】通过三亲杂交和同源重组,在rhcN起始密码子ATG下游插入了一个Km抗性基因,利用PCR扩增和Southern blot对插入情况进行验证,从而构建rhcN插入突变体HH103ΩrhcN。利用大豆染料木苷（Genistin）对野生型根瘤菌HH103及HH103ΩrhcN突变体进行诱导,并对处理组合对照组进行胞外蛋白纯化,通过Western blot检测rhcN突变对根瘤菌III型效应因子NopC及NopT分泌的影响。利用双层钵植物培养系统对大豆Williams82进行野生型根瘤菌HH103和HH103ΩrhcN突变体接种,分析rhcN突变对根瘤表型的影响。利用前期收集的100份基因型差异的大豆品种进行结瘤能力鉴定,分析根瘤菌Ⅲ型效应因子分泌异常情况下在不同基因型大豆品种中的结瘤表现。【结果】通过PCR扩增和Southern blot验证了成功构建的HH103ΩrhcN突变体,胞外蛋白鉴定表明rhcN突变能够抑制根瘤菌Ⅲ型效应因子NopC和NopT分泌。利用HH103ΩrhcN突变体和野生型HH103在Williams82进行结瘤鉴定,结果表明,rhcN突变能够显著抑制根瘤数和根瘤干重。对100份基因型差异的大豆品种进行结瘤鉴定,结果表明,rhcN突变能够引起其中80份大豆资源根瘤数目降低,13份根瘤数目显著升高,7份不发生显著性变化。【结论】rhcN突变能够引起根瘤菌Ⅲ型效应因子的异常分泌,进而能够影响共生体系的建立。根瘤菌III型效应因子在大豆-根瘤菌共生体系形成过程中发挥着重要作用,并且不同基因型大豆遗传背景对根瘤菌Ⅲ型效应因子具有不同应对反应。

关键词: 大豆, 根瘤菌, III型效应因子, rhcN, 共生

Abstract:

【Background】 Soybean is an important economic crop, which is the main source of food and feed. Symbiosis is a special character of soybean to fix nitrogen in air and transferred into ammonia via rhizobia. Via symbiosis soybean can acquire enough nitrogen source for development. Rhizobial type Ⅲ effectors play pivotal roles in regulating the establishment of symbiosis. The rhcN gene codes an ATP transferase, which can regulate the secretion of type Ⅲ effector of rhizobium into host cells. 【Objective】To elucidate the symbiotic mechanism will help to improve the nitrogen fixation efficiency of soybean-rhizobium symbiosis system and is useful for friendly agricultural development. 【Method】In this study, rhcN mutant was constructed by tri-parental mating method and inserted a kanamycin gene in the downstream of rhcN start codon. The mutant was confirmed by PCR and Southern blot. To detect whether the expression of other type Ⅲ effectors was affected by rhcN mutant. The expression of NopC and NopT were identified by induced by genistein via Western blot method. Nodulation experiment were performed in Leonard jars, to detect the nodule phenotype of Williams82 after inoculation with the wild strain HH103 and derived HH103ΩrhcN mutant. Finally, one-hundred soybean germplasms with different genotypes were used for nodulation experiment. 【Result】The results of PCR and Southern blot confirmed that HH103ΩrhcN mutant was successful, and the extracellular protein identification supported that rhcN mutant can inhibit the secretion of type Ⅲ effectors NopC and NopT. The nodulation test of Williams82 showed that rhcN mutation can significantly inhibit the nodule number and dry weight. Using HH103ΩrhcN mutant and wild type HH103, 100 core germplasm accessions were identified, these results showed that rhcN mutation could reduce the root nodule phenotype of 80 soybean accessions, significantly increase the root nodule phenotype of 13 soybean accessions, and 7 soybean resources did not change significantly. 【Conclusion】The results showed that the abnormal secretion of type Ⅲ effector factors of rhizobium could affect the establishment of symbiotic system. rhcN played an important role in the formation of soybean rhizobium symbiosis system, and different soybean genetic background had different responses.

Key words: soybean, rhizobium, type Ⅲ effector, rhcN, symbiosis

刘函西,吕浩,郭广雨,刘冬旭,石岩,孙志君,张泽鑫,张艳娇,文莹楠,王洁琦,刘春燕,陈庆山,辛大伟,王锦辉. 大豆根瘤菌HH103 rhcN突变对结瘤能力的影响[J]. 中国农业科学, 2021, 54(6): 1104-1111.

HanXi LIU,Hao LÜ,GuangYu GUO,DongXu LIU,Yan SHI,ZhiJun SUN,ZeXin ZHANG,YanJiao ZHANG,YingNan WEN,JieQi WANG,ChunYan LIU,QingShan CHEN,DaWei XIN,JinHui WANG. Effect of rhcN Gene Mutation on Nodulation Ability of Soybean Rhizobium HH103[J]. Scientia Agricultura Sinica, 2021, 54(6): 1104-1111.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2021.06.003

https://www.chinaagrisci.com/CN/Y2021/V54/I6/1104

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参考文献 35

[1]	沈琼, 刘小和. 我国油料、植物油的进口特征及品种间的替代性分析. 中国农村经济, 2006(5):25-31.
	SHEN Q, LIU X H. Analysis on the import characteristics of my country's oilseeds and vegetable oils and the substitution among varieties. China's Rural Economy, 2006(5):25-31. (in Chinese)
[2]	魏培梅. 国际大豆供求背景下中国大豆出口贸易现状及对策. 对外经贸实务, 2019(9):45-49.
	WEI P M. China's soybean export trade status and countermeasures under the background of international soybean supply and demand. Foreign Economic and Trade Practices, 2019(9):45-49. (in Chinese)
[3]	马利平. 生物有机肥替代化肥减施对大豆土壤微生物多样性的影响[D]. 哈尔滨: 黑龙江大学, 2019.
	MA L P. Effects of reduced application of bio-organic fertilizer instead of chemical fertilizer on soybean soil microbial diversity[D]. Harbin: Heilongjiang University, 2019. (in Chinese)
[4]	FOLEY J A, RAMANKUTTY N, BRAUMAN K A, CASSIDY E S, GERBER J S, JOHNSTON M, MUELLER N D, CONNELL C O, RAY D K, WEST P C, BALZER C, BENNETT E M, CARPENTER S R, HILL J, MONFREDA C, POLASKY S, ROCKSTRÖM J, SHEEHAN J, SIEBERT S, TILMAN D, ZAKS D P. Solutions for a cultivated planet. Nature, 2011,478(7369):337-342. doi: 10.1038/nature10452 pmid: 21993620
[5]	ROCKSTROM J, STEFFEN W, NOONE K, PERSSON A, CHAPIN F S, LAMBIN E F, LENTON T M, SCHEFFER M, FOLKE C, SCHELLNHUBER H J, NYKVIST B, DE WIT C A, HUGHES T, DER LEEUW S V, RODHE H, SORLIN S, SNYDER P K, COSTANZA R, SVEDIN U, FALKENMARK M, KARLBERG L, CORELL R W, FABRY V J, HANSEN J, WALKER B, LIVERMAN D, RICHARDSON K, CRUTZEN P, FOLEY J A. A safe operating space for humanity. Nature, 2009,461(7263):472-475. doi: 10.1038/461472a pmid: 19779433
[6]	CONTADOR CAROLINA A, LO SIU-KIT, CHAN SIU H J, LAM H M. Metabolic analyses of nitrogen fixation in the soybean microsymbiont Sinorhizobium fredii using constraint-based modeling. Msystems, 2020,5(1):e00516-e00519. doi: 10.1128/mSystems.00516-19 pmid: 32071157
[7]	WILLEMS A, COLLINS M D. Phylogenetic analysis of Rhizobia and Agrobacteria based on 16S rRNA gene sequences. International Journal of Systematic Bacteriology, 1993,43(2):305-313. doi: 10.1099/00207713-43-2-305 pmid: 8494742
[8]	PEOPLES M B, HERRIDGE D F, LADHA J K. Biological nitrogen fixation: An efficient source of nitrogen for sustainable agricultural production. Plant and Soil, 1995,174(1/2):3-28. doi: 10.1007/BF00032239
[9]	KIERS E T, HUTTON M G, DENISON R F. Human selection and the relaxation of legume defenses against ineffective rhizobia. Proceedings of the Royal Society B, 2007,274(1629):3119-3126.
[10]	HU Y, HUANG H, CHENG X, SHU X, WHITE A P, STAVRINIDES J, KOSTER W, ZHU G, ZHAO Z, WANG Y. A global survey of bacterial type III secretion systems and their effectors. Environmental Microbiology, 2017,19(10):3879-3895. doi: 10.1111/1462-2920.13755 pmid: 28401683
[11]	HABENSTEIN B, EI MAMMERI N, TOLCHARD J, LAMON G, TAWANI A, BERBON M, LOQUET A. Structures of type III secretion system needle filaments. Current Topics in Microbiology and Immunology, 2020(427):109-131.
[12]	MARTIN K, ALLAN D J. Identification of protein secretion systems and novel secreted proteins in Rhizobium leguminosarum bv. viciae. BMC Genomics, 2008,9(1):55. doi: 10.1186/1471-2164-9-55
[13]	KIM W S, KRISHNAN H B. A nopA deletion mutant of Sinorhizobium fredii USDA257, a soybean symbiont, is impaired in nodulation. Current Microbiology, 2014,68(2):239-246. doi: 10.1007/s00284-013-0469-4 pmid: 24121614
[14]	LORIO J C, KIM W S, KRISHNAN H B. NopB, a soybean cultivar-specificity protein from Sinorhizobium fredii USDA257, is a type III secreted protein. Molecular Plant-Microbe Interactions, 2004,17(11):1259-1268. pmid: 15553251
[15]	MARIE C, DEAKIN W J, VIPREY V, KOPCINSKA J, GOLINOWSKI W, KRISHNAN H B, PERRET X, BROUGHTON W J. Characterization of Nops, nodulation outer proteins, secreted via the type III secretion system of NGR234. Molecular Plant-Microbe Interactions, 2003,16(9):743-751. doi: 10.1094/MPMI.2003.16.9.743 pmid: 12971597
[16]	TAMPAKAKI A P. Commonalities and differences of T3SSs in rhizobia and plant pathogenic bacteria. Frontier in Plant Science, 2014,5:114.
[17]	KRISHNAN H B, LORIO J, KIM W S, JIANG G, KIM K Y, DEBOER M, PUEPPKE S G. Extracellular proteins involved in soybean cultivar-specific nodulation are associated with pilus-like surface appendages and exported by a type III protein secretion system in Sinorhizobium fredii USDA257. Molecular Plant-Microbe Interactions, 2003,16(7):617-625. doi: 10.1094/MPMI.2003.16.7.617 pmid: 12848427
[18]	VIPREY V, DEL GRECO A, GOLINOWSKI W, BROUGHTON W J, PERRET X. Symbiotic implications of type III protein secretion machinery in Rhizobium. Molecular Microbiology, 1998,28(6):1381-1389. doi: 10.1046/j.1365-2958.1998.00920.x pmid: 9680225
[19]	JIMÉNEZ-GUERRERO I, PÉREZ-MONTAO F, CARLOS M, JAVIER O F, LÓPEZ-BAENA F J, PETER M. NopC is a rhizobium- specific type 3 secretion system effector secreted by sinorhizobium (ensifer) fredii HH103. PLoS ONE, 2015,10(11):e0142866. doi: 10.1371/journal.pone.0142866 pmid: 26569401
[20]	LOPEZBAENA F J, MONREAL J A, PEREZMONTANO F, GUASCHVIDAL B, BELLOGIN R A, VINARDELL J M, OLLERO F J. The absence of Nops secretion in Sinorhizobium fredii HH103 increases GmPR1 expression in williams soybean. Molecular Plant-Microbe Interactions, 2009,22(11):1445-1454. doi: 10.1094/MPMI-22-11-1445 pmid: 19810813
[21]	SKORPIL P, SAAD M M, BOUKLI N M, KOBAYASHI H, ARES ORPEL F, BROUGHTON W J, DEAKIN W J. NopP, a phosphory-lated effector of Rhizobium sp. strain NGR234, is a major determinant of nodulation of the tropical legumes Flemingia congesta and Tephrosia vogelii. Molecular Microbiology, 2005; 57:1304-1317. doi: 10.1111/j.1365-2958.2005.04768.x pmid: 16102002
[22]	朱华晨, 许新萍, 李宝健. 一种简捷的Southern印迹杂交方法. 中山大学学报(自然科学版), 2004(4):128-130.
	ZHU H C, XU X P, LI B J. A simple southern blot hybridization method. Journal of Sun Yat-sen University (Natural Science Edition), 2004(4):128-130. (in Chinese)
[23]	龙火生, 杨公社, 庞卫军. 一种改进的Western杂交方法. 畜牧兽医杂志, 2003(3):11-13.
	LONG H S, YANG G S, PANG W J. An improved western hybridization method. Journal of Animal Husbandry and Veterinary Medicine, 2003(3):11-13. (in Chinese)
[24]	李志辉, 傅豪, 靳巧玲, 段红光, 戴晋, 吴鹤敏. 大豆日光温室加代应用研究初报. 河北农业科学, 2017,21(2):88-91.
	LI Z H, FU H, JIN Q L, DUAN H G, DAI J, WU H M. Preliminary report on the application of soybean solar greenhouse. Hebei Agricultural Sciences, 2017,21(2):88-91. (in Chinese)
[25]	于妍, 刘芳, 唐敬仙. 大豆生育期类型划分研究进展. 北京农业, 2015(8):27.
	YU Y, LIU F, TANG J X. Research progress on classification of soybean growth period. Beijing Agriculture, 2015(8):27. (in Chinese)
[26]	KIMBREL J A, THOMAS W J, JIANG Y, CREASON A L, THIREAULT C A, SACHS J L, CHANG J H. Mutualistic co-evolution of type iii effector genes in sinorhizobium fredii and bradyrhizobium japonicum. PLoS Pathogens, 2013,9(2):e1003204. pmid: 23468637
[27]	LÓPEZ-BAENA F, RUIZ-SAINZ J, RODRÍGUEZ-CARVAJAL M, VINARDELL J. Bacterial molecular signals in the Sinorhizobium fredii-soybean symbiosis. International Journal of Molecular Sciences, 2016,17(5):755. doi: 10.3390/ijms17050755
[28]	JIMÉNEZ-GUERRERO I, PEREZ-MONTANO, ZDYB A, BEUTLER M, WERNER G, GOTTFERT M, JAVIER OLLERO F, MARIA YINARDELL J, LOPEZ-BAENA F J. GunA of Sinorhizobium (Ensifer) fredii HH103 is a T3SS-secreted cellulase that differentially affects symbiosis with cowpea and soybean. Plant and Soil, 2019,435(1/2):15-26. doi: 10.1007/s11104-018-3875-3
[29]	TRIPLETT E W, SADOWSKY M J. Genetics of competition for nodulation of legumes. Annual Review of Microbiology, 1992,46:399-428. doi: 10.1146/annurev.mi.46.100192.002151 pmid: 1444262
[30]	HWANG S, RAY J D, CREGAN P B, ANDY KING C, DAVIES M K, PURCELL L C. Genetics and mapping of quantitative traits for nodule number, weight, and size in soybean (Glycine max L. [Merr.]). Euphytica, 2014,195(3):419-434. doi: 10.1007/s10681-013-1005-0
[31]	YANG S, TANG F, GAO M, KRISHNAN H B, ZHU H. R gene-controlled host specificity in the legume-rhizobia symbiosis. Proceedings of the National Academy of Sciences of the USA, 2010,107(43):18735-18740.
[32]	TANG F, YANG S, LIU J, ZHU H. Rj4, a gene controlling nodulation specificity in soybeans, encodes a thaumatin-like protein but not the one previously reported. Plant Physiology, 2016,170(1):26-32. doi: 10.1104/pp.15.01661 pmid: 26582727
[33]	SUGAWARA M, TAKAHASHI S, UMEHARA Y, LWANO H, TSURUMARU H, ODAKE H, SUZUKI Y, KONDO H, KONNO Y, YAMAKAWA T, SATO S, MITSUI H, MINAMISAWA K. Variation in bradyrhizobial NopP effector determines symbiotic incompatibility with Rj2-soybeans via effector-triggered immunity. Nature Communications, 2018,9(1):3139. pmid: 30087346
[34]	FARUQUE O M, MIWA H, YASUDA M, FUJII Y, KANEKO T, SATO S, OKAZAKI S. Identification of Bradyrhizobium elkanii genes involved in incompatibility with soybean plants carrying the Rj4 allele. Applied and Environmental Microbiology, 2015,81(19):6710-6717. doi: 10.1128/AEM.01942-15 pmid: 26187957
[35]	ZHANG Y, LIU X, CHEN L, FU Y, LI C, QI Z, ZOU J, ZHU R, LI S, WEI W, WANG J, CHANG H, SHI Y, WANG J, LI Q, ZHU J, LI J, JIANG H, WU X, JIA C, YIN Z, HU Z, LIU C, CHEN Q, XIN D. Mining for genes encoding proteins associated with NopL of Sinorhizobium fredii HH103 using quantitative trait loci in soybean (Glycine max Merr.) recombinant inbred lines. Plant and Soil, 2018,431(1/2):245-255. doi: 10.1007/s11104-018-3745-z

参数 Parameter	HH103		HH103ΩrhcN
参数 Parameter	根瘤数目 NN	根瘤干重 NDW (mg)	根瘤数目 NN	根瘤干重 NDW (mg)
平均值 Average	35.52±5.64	34.37±6.35	17.04±10.56**	16.32±11.55**
最大值 Maximum	49.60±4.67	50.84±7.49	45.20±4.87	47.68±6.58
最小值 Minimum	11.50±4.84	12.98±3.65	3.00±0.87	0.35±0.26
标准差 Standard deviation	5.64	6.35	10.56	11.55
变异系数 Coefficient of Variation	54.24	61.02	101.57	111.07