Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (6): 1104-1111.doi: 10.3864/j.issn.0578-1752.2021.06.003

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Effect of rhcN Gene Mutation on Nodulation Ability of Soybean Rhizobium HH103

HanXi LIU1(),Hao LÜ1,GuangYu GUO2,DongXu LIU1,Yan SHI1,ZhiJun SUN1,ZeXin ZHANG1,YanJiao ZHANG1,YingNan WEN1,JieQi WANG1,ChunYan LIU1,QingShan CHEN1(),DaWei XIN1(),JinHui WANG1()   

  1. 1College of Agriculture, Northeast Agricultural University, Harbin 150030
    2Harbin Academy of Agricultural Science, Harbin 150028
  • Received:2020-09-03 Accepted:2020-10-26 Online:2021-03-16 Published:2021-03-25
  • Contact: QingShan CHEN,DaWei XIN,JinHui WANG E-mail:396525797@qq.com;qshchen@126.com;dwxin@neau.edu.com;jinhuiwang113@126.com

RichHTML

39

PDF

699

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

Fig. 1

PCR analysis of HH103ΩrhcN M: Trans 2K Plus DNA marker; 1: HH103ΩrhcN（rhcN-F,rhcN-R）; 2: HH103（rhcN-F,rhcN-R）; 3: HH103ΩrhcN（rhcN-F,Km-SpeⅠ-R）; 4: HH103（rhcN-F,Km-SpeⅠ-R）"

Fig. 2

Southern blot identification of HH103ΩrhcN 1: The genome of HH103ΩrhcN was digested by HindⅢ; 2: The genome of the wild strain HH103 was digested by HindⅢ; 3: The genome of HH103ΩrhcN was digested by SacⅠ; 4: The genome of the wild strain HH103 was digested by SacⅠ"

Fig. 3

Detection of NopC and NopT in secreted proteins +: The presence of Genistein; -: The absence of Genistein"

Fig. 4

Root morphology and nodules traits after HH103 and HH103ΩrhcN inoculation The left picture showed the information of soybean roots and nodules inoculated with HH103, and the right picture showed the information of soybean roots and nodules inoculated with HH103Ωrhcn"

Fig. 5

Nodule number and dry weight of HH103 and HH103ΩrhcN inoculation *for the difference was significant at P<0.05 level"

Table 1

Analysis of nodule traits in 100 germplasm accessions"

参数
Parameter		 HH103 HH103ΩrhcN
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
[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
[1] LI YongJuan, ZHANG YueTong, WANG YiBo, ZHAO ChangJiang, SONG Jie, CHEN XueLi, YAO Qin. Effects of Biochar Application on the Abundance and Community Composition of Nitrogen-Fixing Microbial nifH Gene in Soybean Rotation and Continuous Cropping Systems [J]. Scientia Agricultura Sinica, 2026, 59(6): 1272-1285.
[2] LIU FangDong, SUN Lei, WANG WuBin, ZHAO JinMing, GAI JunYi. Changes of Cropping System and Suggestions on Ecological Cultivation Regions of Soybeans in China [J]. Scientia Agricultura Sinica, 2026, 59(3): 486-498.
[3] CAI TingYang, ZHU YuPeng, LI RuiDong, WU ZongSheng, XU YiFan, SONG WenWen, XU CaiLong, WU CunXiang. Effects of Leaf-Cutting at Seedling Stage on Photosynthetic Characteristics, Pod Distribution and Yield Formation in Soybean in the Huang-Huai-Hai Region [J]. Scientia Agricultura Sinica, 2026, 59(2): 292-304.
[4] WU Qiong, XIE XiangTing, WANG Lei, MOU Yong, LI JinWei. Development and Validation of Event-Specific PCR Method for the Quantification of Genetically Modified Soybean DBN8205 [J]. Scientia Agricultura Sinica, 2026, 59(1): 29-40.
[5] LIU LuPing, HU XueJie, QI Jin, CHEN Qiang, LIU Zhi, ZHAO TianTian, SHI XiaoLei, LIU BingQiang, MENG QingMin, ZHANG MengChen, HAN TianFu, YANG ChunYan. Cloning of the Promoters and Analysis of Expression Patterns of Maturity Genes E1 and E2 in Soybean [J]. Scientia Agricultura Sinica, 2025, 58(5): 840-850.
[6] ZHENG Yu, CHEN Yi, TI JinSong, SHI LongFei, XU XiaoBo, LI YuLin, GUO Rui. Evaluation of Carbon Footprint and Economic Benefit of Different Tobacco Rotation Patterns [J]. Scientia Agricultura Sinica, 2025, 58(4): 733-747.
[7] ZHANG Qi, XUE FuZhen, YANG XiuJie, JIANG SuYang, HUANG XueJuan, MA JiaYi, ZHANG ZheWen, XU JieFei. Study on the Function of Soybean Nicotinamide Enzyme GmNIC1 Gene Under Saline Alkali Stress [J]. Scientia Agricultura Sinica, 2025, 58(24): 5128-5142.
[8] MA HeXiao, GE GuoLong, ZHANG XiangQian, LU ZhanYuan, WANG ManXiu, RONG MeiRen, SHI JingJing, ZHANG DeJian, SUN XuePing. Effects of Different Crop Rotation Systems on Soil Readily Oxidized Organic Carbon and Carbon Pool Activity Differences [J]. Scientia Agricultura Sinica, 2025, 58(24): 5201-5215.
[9] GAO ChunHua, ZHAO HaiJun, ZHAO FengTao, KONG WeiLin, JU FeiYan, LI ZongXin, SHI DeYang, LIU Ping. Effect of Growth Regulators on the Stem Characteristics and Yield of Summer Maize in Maize-Soybean Strip Intercropping [J]. Scientia Agricultura Sinica, 2025, 58(23): 4920-4935.
[10] YANG ShuQi, ZHAO YingXing, QIAN Xin, ZHANG XuePeng, MENG WeiWei, SUI Peng, LI ZongXin, CHEN YuanQuan. Comprehensive Evaluation of the Maize-Soybean Intercropping Pattern in the Huang-Huai Region [J]. Scientia Agricultura Sinica, 2025, 58(23): 4936-4951.
[11] FANG Jian, QIN ZhaoJi, YU YuanYuan, YU NingNing, ZHAO Bin, LIU Peng, REN BaiZhao, ZHANG JiWang. Impacts of Varying Row Ratio Arrangements on Plant Performance, Stand Yield, and Comprehensive Benefits in Soybean-Maize Strip intercropping [J]. Scientia Agricultura Sinica, 2025, 58(23): 4841-4857.
[12] SONG XuHui, ZHAO XueYing, ZHAO Bin, REN BaiZhao, ZHANG JiWang, LIU Peng, REN Hao. Effects of Row Ratio Allocation on Light Distribution and Photosynthetic Production Capacity of Maize-Soybean Strip Intercropping [J]. Scientia Agricultura Sinica, 2025, 58(23): 4858-4871.
[13] SHI DeYang, GAO ChunHua, LI YanHong, ZHAO HaiJun, XIA DeJun. Effects of Row Spacing Configuration on the Canopy Characteristics and Grain Yield of the Intercropping Maize [J]. Scientia Agricultura Sinica, 2025, 58(23): 4872-4885.
[14] ZHANG MengYu, HE ZaiJu, WANG XingXing, REN Hao, REN BaiZhao, LIU Peng, ZHANG JiWang, ZHAO Bin. The Influences of Different Plant Height Combinations of Maize Varieties on Light Distribution in the Canopy and the Photosynthetic Characteristics of Maize Under Maize-Soybean Strip Intercropping Pattern [J]. Scientia Agricultura Sinica, 2025, 58(23): 4886-4904.
[15] KONG WeiLin, GAO ChunHua, ZHAO FengTao, JU FeiYan, LI ZongXin, ZHAO HaiJun, LIU Ping. Effects of Nitrogen Application Rate Combined with Drip Irrigation Amount After Sowing on Yield, Economic Benefit, Water Use Characteristics of Maize-Soybean Strip Intercropping Planting System [J]. Scientia Agricultura Sinica, 2025, 58(23): 4905-4919.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd