贝莱斯芽孢杆菌LJ02中植物免疫蛋白的筛选及其功能

doi:10.3864/j.issn.0578-1752.2021.16.008

摘要/Abstract

摘要：

【目的】贝莱斯芽孢杆菌（Bacillus velezensis）LJ02菌株能够诱导黄瓜等植物产生免疫抗病反应。本研究从生防菌LJ02中分离鉴定具有激发植物免疫活性的蛋白分子,通过验证其功能,进一步解析免疫蛋白的免疫信号通路。【方法】用硫酸铵沉淀法处理LJ02菌株发酵液,离心获得LJ02蛋白粗提物,将其经凝胶层析后再利用半制备高效液相色谱（high performance liquid chromatography,HPLC）分离,收集不同峰值处的蛋白组分。以烟草花叶病毒（tobacco mosaic virus,TMV）为靶标进行活性组分的筛选,从而获得具有免疫活性的组分。液相质谱联用仪（LC-MS）检测分析活性组分后发现,在组分F-23中含鞭毛蛋白（flagellin,Flg^LJ02）。将鞭毛蛋白Flg^LJ02进行原核表达并纯化,经过敏性坏死反应（hypersensitive reaction,HR）和抗病试验验证其免疫功能。为确定其主要免疫信号通路,采用实时荧光定量PCR（qRT-PCR）检测水杨酸（salicylic acid,SA）和乙烯（ethylene,ET）途径的相关合成酶基因ICS1、PAL、ACS1以及免疫相关抗性基因NPR1、PR-1、EIN2和EIN3,通过免疫相关抗性基因的相对表达量解析鞭毛蛋白Flg^LJ02免疫信号转导途径。【结果】HPLC层析分离出贝莱斯芽孢杆菌LJ02菌液产生的60个外泌蛋白组分（F-1—F-60）,免疫烟草接种TMV后,观察分析发现组分F-20、F-23、F-41、F-44具有显著的免疫抗病效应,其中组分F-23抗TMV效果最为显著,其抑制率为81.7%。经过质谱数据分析发现该组分包含鞭毛蛋白Flg^LJ02等7种物质。选择鞭毛蛋白Flg^LJ02为研究对象构建表达载体,转化大肠杆菌BL21,诱导表达后破碎细胞,获得粗提蛋白。将粗提蛋白过柱、透析纯化后的Flg^LJ02渗入珊西烟（Nicotiana tabacum var. xanthi）叶片,24 h后能观察到过敏性坏死反应。将Flg^LJ02稀释为终浓度50、100、200 μg·mL ^-1的稀释液后预处理珊西烟叶片,24 h后接种TMV,其对TMV的枯斑抑制率分别为65.6%、76.1%、88.1%。用鞭毛蛋白Flg^LJ02处理珊西烟,其SA途径和ET信号途径的免疫抗病相关基因ICS1、PAL、NPR1、PR-1、ACS1、EIN2、EIN3的相对表达量在24—48 h均显著上调。【结论】贝莱斯芽孢杆菌LJ02外泌的鞭毛蛋白Flg^LJ02可以激发珊西烟植株的免疫防御反应,提高烟草植株对TMV的抗病性。

关键词: 贝莱斯芽孢杆菌, 鞭毛蛋白, 免疫蛋白, 珊西烟, 烟草花叶病毒

Abstract:

【Objective】Bacillus velezensis LJ02 induces the immune response of cucumbers and other crops. The objective of this study is to screen and identify the immune proteins of LJ02, and further analyze the immune signalling pathways by verifying their functions.【Method】The LJ02 fermentation broth was precipitated with ammonium sulfate precipitation method, and the crude proteins of LJ02 were obtained by centrifugation. Then crude proteins were gel chromatographed and separated by high performance liquid chromatography (HPLC) to collect protein components at different peaks. The immune components were tested with tobacco mosaic virus (TMV) to obtain the immune components in plant. Liquid-phase mass spectrometry (LC-MS) detection and analysis revealed that the component F-23 contained flagellin (Flg^LJ02). Purified recombinant protein Flg^LJ02 produced from Escherichia coli expression system was infiltrated into tobacco leaves and its immune function was verified by hypersensitive reaction (HR) and immune resistance analysis. Real-time fluorescent quantitative PCR (qRT-PCR) was used to detect salicylic acid (SA) and ethylene (ET) key synthetase genes ICS1, PAL, ACS1 and immune-related resistance genes NPR1, PR-1, EIN2 and EIN3. The relative expression of its immune-related resistance genes was tested to identify the Flg ^LJ02-induced immune signal transduction pathway.【Result】Sixty exocrine protein components (from F-1 to F-60) of LJ02 were separated from HPLC, and components F-20, F-23, F-41, F-44 had strong immune effect against TMV in tobacco, among which, component F-23 showed the most significant anti-TMV effect, with an inhibition rate of 81.7%. Further mass spectrometry analysis found that this component contained flagellin Flg^LJ02 and other 6 substances. The Flg^LJ02expression vector was constructed, and then transformed into E. coli BL21, the cells were broken after inducing the expression of Flg ^LJ02. The crude protein was eluted with Ni column purification, further dialyzed, then injected Flg^LJ02 into tobacco leaves, hypersensitive reactions appeared about 24 h. Tobacco leaves was infiltrated with 50, 100 and 200 μg·mL ^-1 Flg^LJ02, and then TMV was inoculated 24 hours post Flg^LJ02inoculation (hpi). The inhibition rate of Flg^LJ02against TMV was 65.6%, 76.1% and 88.1%, respectively. The qRT-PCR determined that the expressions of SA and ET via defense-related genes ICS1, PAL, NPR1, PR-1, ACS1, EIN2, and EIN3 in tobacco plants were significantly up-regulated in 24-48 h.【Conclusion】The Flg ^LJ02 secreted by the B. velezensis LJ02 activates the SA and ET signalling immune pathway, thereby improving plants disease resistance to TMV.

Key words: Bacillus velezensis, flagellin, immune protein, Nicotiana tabacum var. xanthi, tobacco mosaic virus (TMV)

魏艳侠,李卓然,张斌,苑瑜瑾,于玮玮,常若葵,王远宏. 贝莱斯芽孢杆菌LJ02中植物免疫蛋白的筛选及其功能[J]. 中国农业科学, 2021, 54(16): 3451-3460.

WEI YanXia,LI ZhuoRan,ZHANG Bin,YUAN YuJin,YU WeiWei,CHANG RuoKui,WANG YuanHong. Screening and Function of Plant Immune Proteins from Bacillus velezensis LJ02[J]. Scientia Agricultura Sinica, 2021, 54(16): 3451-3460.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2021.16.008

https://www.chinaagrisci.com/CN/Y2021/V54/I16/3451

图/表 8

表1

图1

表2

图2

图3

图4

图5

图6

参考文献 44

[1]	RABBEE M F, ALI M S, CHOI J, HWANG B S, JEONG S C, BAEK K H. Bacillus velezensis: A valuable member of bioactive molecules within plant microbiomes. Molecules, 2019, 24(6):1046. doi: 10.3390/molecules24061046
[2]	RAHMAN A, UDDIN W, WENNER N G. Induced systemic resistance responses in perennial ryegrass against Magnaporthe oryzae elicited by semi-purified surfactin lipopeptides and live cells of Bacillus amyloliquefaciens. Molecular Plant Pathology, 2015, 16(6):546-558. doi: 10.1111/mpp.2015.16.issue-6
[3]	FAN B, BLOM J, KLENK H P, BORRISS R. Bacillus amyloliquefaciens, Bacillus velezensis, and Bacillus siamensis form an “Operational Group B. amyloliquefaciens” within the B. subtilis species complex. Frontiers in Microbiology, 2017, 8:22.
[4]	FAN B, WANG C, SONG X F, DING X L, WU L M, WU H J, GAO X F, BORRISS R. Bacillus velezensis FZB42 in 2018: The Gram-positive model strain for plant growth promotion and biocontrol. Frontiers in Microbiology, 2018, 9:2491. doi: 10.3389/fmicb.2018.02491
[5]	PANDIN C, DARSONVAL M, MAYEUR C, LE COQ D, AYMERICH S, BRIANDET R. Biofilm formation and synthesis of antimicrobial compounds by the biocontrol agent Bacillus velezensis QST713 in an Agaricus bisporus compost micromodel. Applied and Environmental Microbiology, 2019, 85(12):e00327-19.
[6]	CHEN M, WANG J, LIU B, ZHU Y, XIAO R, YANG W, GE C, CHEN Z. Biocontrol of tomato bacterial wilt by the new strain Bacillus velezensis FJAT-46737 and its lipopeptides. BMC Microbiology, 2020, 20(1):160. doi: 10.1186/s12866-020-01851-2
[7]	WANG N B, LIU M J, GUO L H, YANG X F, QIU D W. A novel protein elicitor (PeBA1) from Bacillus amyloliquefaciens NC6 induces systemic resistance in tobacco. International Journal of Biological Sciences, 2016, 12(6):757-767. doi: 10.7150/ijbs.14333
[8]	RANF S, GISCH N, SCHAFFER M, ILLIG T, WESTPHAL L, KNIREL Y A, SANCHEZ-CARBALLO P M, ZAHRINGER U, HUCKELHOVEN R, LEE J, SCHEEL D. A lectin S-domain receptor kinase mediates lipopolysaccharide sensing in Arabidopsis thaliana. Nature Immunology, 2015, 16(4):426-433. doi: 10.1038/ni.3124
[9]	HE P, SHAN L, SHEEN J. Elicitation and suppression of microbe- associated molecular pattern-triggered immunity in plant-microbe interactions. Cellular Microbiology, 2007, 9(6):1385-1396. doi: 10.1111/cmi.2007.9.issue-6
[10]	NEWMAN M A, SUNDELIN T, NIELSEN J T, ERBS G. MAMP (microbe-associated molecular pattern) triggered immunity in plants. Frontiers in Plant Science, 2013, 4:139.
[11]	NAVEED Z A, WEI X, CHEN J, MUBEEN H, ALI G S. The PTI to ETI continuum in Phytophthora-plant interactions. Frontiers in Plant Science, 2020, 11:593905. doi: 10.3389/fpls.2020.593905
[12]	KUMAR D. Salicylic acid signaling in disease resistance. Plant Science, 2014, 228:127-134. doi: 10.1016/j.plantsci.2014.04.014
[13]	SUMAYO M S, SON J S, GHIM S Y. Exogenous application of phenylacetic acid promotes root hair growth and induces the systemic resistance of tobacco against bacterial soft-rot pathogen Pectobacterium carotovorum subsp. carotovorum. Functional Plant Biology, 2018, 45(11):1119-1127. doi: 10.1071/FP17332
[14]	ZHENG X Y, ZHOU M, YOO H, PRUNDA-PAZ J L, SPIVEY N W, KAY S A, DONG X. Spatial and temporal regulation of biosynthesis of the plant immune signal salicylic acid. Proceedings of the National Academy of Sciences of the United States of America, 2015, 112(30):9166-9173.
[15]	BACKER R, NAIDOO S, VAN DEN BERG N. The nonexpressor of pathogenesis-related genes 1 (NPR1) and related family: Mechanistic insights in plant disease resistance. Frontiers in Plant Science, 2019, 10:102. doi: 10.3389/fpls.2019.00102
[16]	LINCOLN J E, SANCHEZ J P, ZUMSTEIN K, GILCHRIST D G. Plant and animal PR1 family members inhibit programmed cell death and suppress bacterial pathogens in plant tissues. Molecular Plant Pathology, 2018, 19(9):2111-2123. doi: 10.1111/mpp.2018.19.issue-9
[17]	FRANKOWSKI K, KESY J, KOTARBA W, KOPCEWICZ J. Ethylene signal transduction pathway. Postepy Biochemii, 2008, 54(1):99-106.
[18]	PETRUZZELL L, CORAGGIO I, LEUBNER-METZGER G. Ethylene promotes ethylene biosynthesis during pea seed germination by positive feedback regulation of 1-aminocyclo-propane-1-carboxylic acid oxidase. Planta, 2000, 211(1):144-149. doi: 10.1007/s004250000274
[19]	ZHANG F, WANG L, QI B, ZHAO B, KO E E, RIGGAN N D, CHIN K, QIAO H. EIN2 mediates direct regulation of histone acetylation in the ethylene response. Proceedings of the National Academy of Sciences of the United States of America, 2017, 114(38):10274-10279.
[20]	WEN X, ZHANG C, JI Y, ZHAO Q, HE W, AN F, JIANG L, GUO H. Activation of ethylene signaling is mediated by nuclear translocation of the cleaved EIN2 carboxyl terminus. Cell Research, 2012, 22(11):1613-1616. doi: 10.1038/cr.2012.145
[21]	AN F, ZHAO Q, JI Y, LI W, JIANG Z, YU X, ZHANG C, HAN Y, HE W, LIU Y, ZHANG S, ECKER J R, GUO H. Ethylene-induced stabilization of ETHYLENE INSENSITIVE3 and EIN3-LIKE1 is mediated by proteasomal degradation of EIN3 binding F-box 1 and 2 that requires EIN2 in Arabidopsis. The Plant Cell, 2010, 22(7):2384-2401. doi: 10.1105/tpc.110.076588
[22]	KIM M, LEE C, PARK J, JEON B Y, HONG M. Crystal structure of Bacillus cereus flagellin and structure-guided fusion-protein designs. Scientific Reports, 2018, 8(1):5814. doi: 10.1038/s41598-018-24254-w
[23]	MOHARI B, THOMPSON M A, TRINIDAD J C, SETAYESHGAR S, FUQUA C. Multiple flagellin proteins have distinct and synergistic roles in Agrobacterium tumefaciens motility. Journal of Bacteriology, 2018, 200(23):e00327-18.
[24]	BERG H C, ANDERSON R A. Bacteria swim by rotating their flagellar filaments. Nature, 1973, 245(5425):380-382. doi: 10.1038/245380a0
[25]	FORSTNERIC V, IVICAK-KOCJAN K, LJUBETIC A, JERALA R, BENCINA M. Distinctive recognition of flagellin by human and mouse toll-like receptor 5. PLoS ONE, 2016, 11(7):e0158894. doi: 10.1371/journal.pone.0158894
[26]	MCNAMARA N, GALLUP M, SUCHER A, MALTSEVA I, MCKEMY D, BASBAUM C. AsialoGM1 and TLR5 cooperate in flagellin-induced nucleotide signaling to activate Erk1/2. American Journal of Respiratory Cell and Molecular Biology, 2006, 34(6):653-660. doi: 10.1165/rcmb.2005-0441OC
[27]	VANTHANA M, NAKKEERAN S, MALATHI V G, RENUKADEVI P, VINODKUMAR S. Induction of in planta resistance by flagellin (Flg) and elongation factor-TU (EF-Tu) of Bacillus amyloliquefaciens (VB7) against groundnut bud necrosis virus in tomato. Microbial Pathogenesis, 2019, 137:103757. doi: 10.1016/j.micpath.2019.103757
[28]	刘秀霞, 梁宇宁, 张伟伟, 霍艳红, 冯守千, 邱化荣, 何晓文, 吴树敬, 陈学森. 超表达MdFLS2的拟南芥fls2突变体识别细菌鞭毛蛋白提高对轮纹病菌的抗性. 园艺学报, 2018, 45(5):827-844.
	LIU X X, LIANG Y N, ZHANG W W, HUO Y H, FENG S Q, QIU H R, HE X W, WU S J, CHEN X S. MdFLS2 recognizes bacterial flagellin flg22 and enhances immune resistance against apple ring rot causal fungi in Arabidopsis fls2 mutant. Acta Horticulturae Sinica, 2018, 45(5):827-844. (in Chinese)
[29]	RAJAMANICKAM S, NAKKEERAN S. Flagellin of Bacillus amyloliquefaciens works as a resistance inducer against groundnut bud necrosis virus in chilli (Capsicum annuum L.). Archives of Virology, 2020, 165(7):1585-1597. doi: 10.1007/s00705-020-04645-z
[30]	谷医林, 王远宏, 常若葵, 李宁, 李娟, 徐明珠. 解淀粉芽孢杆菌LJ1诱导黄瓜抗白粉病的研究. 农药学学报, 2013, 15(3):293-298.
	GU Y L, WANG Y H, CHANG R K, LI N, LI J, XU M Z. Characterization of powdery mildew resistance induced by Bacillus amyloliquefaciens LJ1 in cucumber. Chinese Journal of Pesticide Science, 2013, 15(3):293-298. (in Chinese)
[31]	柴庆凯, 张斌, 常若葵, 刘慧芹, 田小卫, 王远宏. 解淀粉芽孢杆菌LJ02对黄瓜抗灰霉病菌的生防效果及其诱导抗性机理的初步研究. 植物病理学报, 2019, 49(6):828-835.
	CHAI Q K, ZHANG B, CHANG R K, LIU H Q, TIAN X W, WANG Y H. Preliminary study on the effect of the induced resistance in cucumber with Bacillus amyloliquefaciens LJ02 against Botrytis cinerea. Acta Phytopatholgica Sinica, 2019, 49(6):828-835. (in Chinese)
[32]	LI Y L, GU Y L, LI J, XU M Z, WEI Q, WANG Y H. Biocontrol agent Bacillus amyloliquefaciens LJ02 induces systemic resistance against cucurbits powdery mildew. Frontiers in Microbiology, 2015, 6:883.
[33]	GOODING G V, HEBERT T T. A simple technique for purification of tobacco mosaic virus in large quantities. Phytopathology, 1967, 57(11):1285.
[34]	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCt method. Methods, 2001, 25(4):402-408. doi: 10.1006/meth.2001.1262
[35]	ALONSO J M, HIRAYAMA T, ROMAN G, NOURIZADEH S, ECKER J R. EIN2, a bifunctional transducer of ethylene and stress responses in Arabidopsis. Science, 1999, 284(5423):2148-2152. doi: 10.1126/science.284.5423.2148
[36]	SOLANO R, STEPANOVA A, CHAO Q, ECHER J R. Nuclear events in ethylene signaling: A transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1. Genes and Development, 1998, 12(23):3703-3714. doi: 10.1101/gad.12.23.3703
[37]	WANG S, HAN K, PENG J, ZHAO J, JIANG L, LU Y, ZHENG H, LIN L, CHEN J, YAN F. NbALD1 mediates resistance to turnip mosaic virus by regulating the accumulation of salicylic acid and the ethylene pathway in Nicotiana benthamiana. Molecular Plant Pathology, 2019, 20(7):990-1004. doi: 10.1111/mpp.2019.20.issue-7
[38]	SHAH C P, KHARKAR P S. Inosine 5′-monophosphate dehydrogenase inhibitors as antimicrobial agents: Recent progress and future perspectives. Future Science Medicinal Chemistry, 2015, 7(11):1415-1429.
[39]	SHUKLA D, HUDA K M, BANU M S, GILL S S, TUTEJA R, TUTEJA N. OsACA6, a P-type 2B Ca²+ ATPase functions in cadmium stress tolerance in tobacco by reducing the oxidative stress load. Planta, 2014, 240(4):809-824. doi: 10.1007/s00425-014-2133-z
[40]	LU Y, SWARTZ J R. Functional properties of flagellin as a stimulator of innate immunity. Scientific Reports, 2016, 6:18379. doi: 10.1038/srep18379
[41]	FELIX G, DURAN J D, VOLKO S, BOLLER T. Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. The Plant Journal, 1999, 18(3):265-276. doi: 10.1046/j.1365-313X.1999.00265.x
[42]	LOPEZ M, MIRANDA E, RAMOS C, GARCIA H, NEIRA- CATTILLO A. Activation of early defense signals in seedlings of Nicotiana benthamiana treated with chitin nanoparticles. Plants, 2020, 9(5):607. doi: 10.3390/plants9050607
[43]	LU D, WU S, GAO X, ZHANG Y, SHAN L, HE P. A receptor-like cytoplasmic kinase, BIK1, associates with a flagellin receptor complex to initiate plant innate immunity. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(1):496-501.
[44]	TSUDA K, SATO M, STODDARD T, GLAZEBROOK J, KATAGIRI F. Network properties of robust immunity in plants. PLoS Genetics, 2009, 5(12):e1000772. doi: 10.1371/journal.pgen.1000772

登录号 ID	基因名称Gene name	正向引物 Forward primer (5′-3′)	反向引物 Reverse primer (5′-3′)
X12737	PR-1	GCTGCTAAGGCCGTTGAGAT	ACATCCAACACGAACCGAGT
AF480488	NPR1	CGGCGGCATGACTGAATTTT	TTAGCGTCGGCGAAGTAGTC
AB289452	PAL	GGAGAACCAAGAACGGTGGT	CTGCCCTTGTCCCTGATTGT
HQ693477	EIN2	CCACCACAAGCAAAACAGGG	GGTTGCCATCTCCACGTCTT
AY740529	ICS1	GCTCGTAGCACCAGAGTTGT	TCCAATGAATGCCGCTGACT
AF247568.1	EIN3	TAATGCGATTCCGGGCAAGA	CCAACGGAAATCGCCTCTGA
NM_001326261.2	ACS1	AGGGCCATTGCCAACTTTCA	AGTTGCACCACCAGCCATAA
U60495	β-actin	ATGCCTATGTGGGTGACGAAG	TCTGTTGGCCTTAGGGTTGAG

名称 Name	序列 Sequence	匹配率 Score (%)	氨基酸数目 Number of amino acid (aa)	蛋白分子量 Molecular weight (kD)
鞭毛蛋白Flagellin (Fragment)	TAIDTVSSER	1.80	306	32.6
肌苷5′-单磷酸脱氢酶Inosine-5′-monophosphate dehydrogenase	VIEFPNSSK	1.79	488	52.9
未知蛋白Uncharacterized protein	LGAESI	1.34	327	36.7
TetR 家族转录调节因子 TetR family transcriptional regulator	VILL	0	385	44.8
P型ATP酶 ATPase P	LAAAAETGSEHPLGEAIVSGAEK	0	811	86.6
未知蛋白Uncharacterized protein	IANDDQINEIIPSEWENVYLYAEIL	0	161	19.2
UPF0348 protein AX282_03230	FLNL	0	415	47.1