微生物学报  2015, Vol. 55 Issue (8): 1010-1017
http://dx.doi.org/10.13343/j.cnki.wsxb.20140546
中国科学院微生物研究所,中国微生物学会,中国菌物学会
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文章信息

李牧原, 徐鹏霞, 张洧琪, 张勇. 2015
Muyuan Li, Pengxia Xu, Weiqi Zhang, Yong Zhang. 2015
青枯菌Rsc1285参与Ⅲ型分泌系统及致病力的调控
Regulation of rsc1285 gene in type Ⅲ secretion system in Ralstonia solanacearum
微生物学报, 201555(8): 1010-1017
Acta Microbiologica Sinica, 201555(8): 1010-1017

文章历史

收稿日期:2014-11-19
修回日期:2015-01-26
青枯菌Rsc1285参与Ⅲ型分泌系统及致病力的调控
李牧原, 徐鹏霞, 张洧琪, 张勇     
西南大学资源环境学院, 重庆 400715
摘要: 【目的】研究青枯菌Rsc1285参与调控其Ⅲ型分泌系统(Type Ⅲ secretion system, T3SS)及致病力的途径。【方法】通过基因敲除、基因互补等研究Rsc1285对T3SS基因表达和致病力的影响。【结果】青枯菌rsc1285基因缺失突变体对寄主西红柿植株的致病力明显减弱,其hrpB、T3SS等基因表达水平较野生型明显降低,但hrpGprhG的表达不受影响。【结论】青枯菌通过一个全新的途径利用Rsc1285调控hrpB及T3SS的转录表达并决定其致病力。
关键词: 青枯菌    Ⅲ型分泌系统    hrp基因簇    致病力    
Regulation of rsc1285 gene in type Ⅲ secretion system in Ralstonia solanacearum
Muyuan Li, Pengxia Xu, Weiqi Zhang, Yong Zhang     
College of Resources and Environment, Southwest University, Chongqing 400715, China
Supported by the National Natural Science Foundation of China (31200067) and by the Basic Scientific Research Foundation of Central Colleges (XDJK2013C156,SWU113016)
Corresponding author. Tel:+ 86-23-68250994; Fax:+86-23-68250109; E-mail: bioyongzhang@gmail.com
Received:19 November 2014/Revised:26 January 2015
Abstract:[Objective] Rsc1285 is one of the putative T3SS-regulated factors in Ralstonia solanacearum, and the regulation of Rsc1285 on T3SS and pathogenicity was characterized. [Methods] The rsc1285 deletion mutants were constructed by homologous recombination and characterized by complementation. [Results] The rsc1285 mutant was significantly less virulent than the wild-type strain to infect tomato plants. Rsc1285 controls the expression of hrpB and HrpB-regulating genes, but it is dispensable for the expression of hrpG and prhG. [Conclusion] R. solanacearum uses Rsc1285 to control the T3SS and pathogenicity via a novel pathway, and this finding provides insights into overall infection mode of R. solanacearum.
Key words: Ralstonia solanacearum    Type Ⅲ secretion system    hrp regulon    pathogenicity    

青枯菌(Ralstonia solanacearum) 是一种革兰氏阴性土传植物病原菌,可侵染50多科450余种植物并导致毁灭性枯萎(bacterial wilt,又称青枯病),是世界上分布最广、危害最严重的十大植物病原细菌之一[1]。和其它革兰氏阴性病原细菌一样,注射器状的III型分泌系统(Type III secretion system,T3SS)是青枯菌的一个决定性致病因子。青枯菌利用T3SS与寄主细胞互作,将各种效应蛋白(Type III effectors,T3Es)注射到寄主细胞中,以抑制寄主的免疫反应,从而引起寄主感病[2, 3]。青枯菌T3SS由hrp(hypersensitive response and pathgenicity)基因簇编码,而hrp基因簇及众多T3Es基因的转录表达受AraC家族转录调控因子HrpB直接控制,HrpB通过对含hrpII框(TTCG-N16-TTCG)启动子的结合控制hrp 基因簇及众多T3Es基因的转录表达,决定致病性[4, 5, 6]。青枯菌hrpB 的转录表达受到OmpR/PhoB家族二组分反应调节子HrpG和PrhG的共同正向调控,其中hrpG的转录表达受到PrhA-PrhI/R-PrhJ信号通路的正调控和LysR家族转录调控因子PhcA的间接负向调控,而prhG的转录表达受PhcA的正调控但和PrhA信号通路无关[8, 9, 10]。基于PhcA的群体效应特性(quorum sensing),Zhang等根据研究结果提出青枯菌可根据自身浓度变化切换HrpG和PrhG动态调控 hrpB及T3SS表达的观点[8]。青枯菌运用精密网络对hrp基因簇及T3SS进行全局性调控,但迄今仍有大量的调控过程有待揭示[3, 4, 11, 12]。为进一步阐明青枯菌hrp基因簇及T3SS表达被调控的分子机理,我们构建了popA-lacZYA融合报告基因以表征青枯菌hrp基因簇的表达水平(popA基因位于hrp基因簇的左侧外端,受HrpB的直接调控,具有和hrp基因簇相同的表达特性,其结构示意图可见[8]),通过构建转座子突变体文库筛选了一批可能影响青枯菌T3SS基因表达的未知因子[8],本文以Rsc1285为研究对象(rsc1285基因位于青枯菌染色体上,编码产物推定和原核细胞染色体分离、染色质凝聚等相关),通过构建rsc1285基因缺失突变体和基因互补等实验,初步研究了Rsc1285对青枯菌hrp基因簇转录表达及致病性的影响,有助于我们从分子水平进一步了解青枯菌致病的全局性调控网络。

1 材料和方法 1.1 材料

1.1.1 供试菌株、质粒及培养条件:本实验所用菌株、质粒等见表1。青枯菌及其衍生菌株均来自OE1-1(生理小种1,生化型4,对番茄和烟草都具有强致病力)[13],大肠杆菌DH5α和S17-1[14]分别用于质粒构建和融合。青枯菌采用B培养液(rich medium)和基础培养液(hrp-inducing medium,又称sucrose medium),在28 ℃条件下振荡培养[15]。大肠杆菌采用LB培养液,37 ℃振荡培养。抗生素使用浓度:氨苄青霉素(Ap) 100 μg/mL;卡那霉素(Km) 50 μg/mL;庆大霉素(Gm) 20 μg/mL;多粘菌素B(Polymyxin B,PB,用于青枯菌培养)50 μg/mL。

表 1 本文所用的菌株及质粒 Table 1. Bacterial strains and plasmids used in the study
Strain or plasmidDescriptionSource or references
strain
E. coli DH5αmcrA φ80 lacZΔM15, recA1,endA1Laboratory stock
E. coli S17-1TpR, SmR, recA, thi, pro, hsdR-M+RP4[15]
R.solanacearum OE1-1wild type,race 1,biovar 4,virulence on tobacco and tomato[13]
RK5050OE1-1 (popA-lacZYA)[17]
RK5046OE1-1 (hrpB-lacZYA)[17]
RK5120OE1-1 (hrpG-lacZYA)[17]
RK5212OE1-1 (prhG-lacZYA)[8]
RK5701RK5050 (Δrsc1285)This study
RK5704RK5046 (Δrsc1285)This study
RK5707RK5120 (Δrsc1285)This study
RK5710RK5212 (Δrsc1285)This study
RK5713RK5701 (rsc1285 complementation)This study
plasmid
pBluescript II KS(+)Amp r, LacZα,Laboratory stock
pK18mobSacBKamr, oriT(RP4), SacB, LacZα[16]
pUC18-miniTn7T-GmGm r, Tn7T, [18]
pTNS2Amp r, T7 transposase expression vector[18]
pK18d1285Kamr, SacB,for rsc1285 gene deletionThis study
pUC1285Gm r, for rsc1285 complementationThis study

1.1.2 主要试剂:PCR聚合酶(Primestar HS polymerase,添加GC-rich buffe、dNTP等)、限制性内切酶(BamH I、Hind III、EcoR I、EcoR V等)、DNA连接酶(DNA Ligation Kit V2.1)等购自大连宝生物(TaKaRa)公司;DNA Marker、DNA染料(Gold View)等购置于天根公司;质粒大、小提试剂盒、琼脂糖凝胶中DNA回收试剂盒、PCR产物纯化试剂盒等购自Omega公司(EZNA系列);所用抗生素、IPTG、X-gal等购自Sigma公司;无机盐试剂、蔗糖、葡萄糖等购自上海生工公司;引物合成及DNA测序由上海生工公司完成。

1.2 青枯菌rsc1285基因缺失突变体的构建

本实验利用基于质粒pK18mobSacB的同源重组技术对目标基因进行敲除(图1)[16]。构建rsc1285基因(1008 bp)缺失质粒时,参照GMI1000全基因组序列(https://iant.toulouse.inra.fr/bacteria/annotation/cgi/ralso.cgi)分别在rsc1285基因的上下游设计成对引物:上游引物对rsc1285A1x (5′-CGTCTAGAGAACCTGCTGATGATGTC-3′,含Xba I酶切位点)和rsc1285B1(5′-TCGCGCGGCAGACA CGGCCGGGTTGAGCGGCTCGTCAT-3′),下游引物对rsc1285A2(5′-ATGACGAGCCGCTCAACCCGGCCGT GTCTGCCGCGCGA-3′)和rsc1285B2H(5′-CGAAGC TTCACGTACTGGAAGACGT-3′,含Hind III酶切位点)。其中,rsc1285B1和rsc1285A2完全互补,各含rsc1285上下游18 bp DNA序列。以青枯菌OE1-1基因组DNA为模板分别PCR扩增rsc1285基因的上下游DNA片段(大小各为约600 bp),PCR扩增条件:98 ℃ 1 min;98 ℃ 10 s,62 ℃ 5 s,72 ℃ 50 s,30个循环;72 ℃ 5 min;4 ℃。PCR产物经琼脂糖凝胶电泳纯化,提取目标DNA片段,将上下游DNA片段混合并用引物对(rsc1285A1X和rsc1285B2H)进行扩增,所得PCR产物(大小约1.2 kb)经琼脂糖凝胶电泳纯化提取。所提DNA经DNA连接酶(DNA Ligation Kit V2.1)作用插入到EcoR V酶切的pBluescript II KS(+)质粒(16 ℃,1 h),然后转化到E. coli DH5α,LB+Ap+X-gal+IPTG平板涂布并挑选白菌落,液体(LB+Ap)培养并提取质粒。提取质粒经BamH I- Hind III双酶切检验合格后,交由上海生工进行序列测定,取测序验证无突变的质粒用于下一步实验,命名为pKSd1285。用BamH I-Hind III双酶切pKSd1285,琼脂糖凝胶电泳分离、提取1.2 kb的插入片段,连接到BamH I-Hind III双酶切的pk18mobsacB质粒中并转化到E coli DH5α,涂布到LB+Km+X-gal+IPTG平板,挑选白菌落,液体(LB+Km)培养并提取质粒。提取的质粒经BamH I-Hind III双酶切、基因测序验证合格后,命名为pK18d1285,用于基因敲除实验。质粒pK18d1285经E. coli S17-1转化、融合到青枯菌中(青枯菌和大肠杆菌混合后用0.45 μm膜过滤),分别经Km和蔗糖筛选后,用引物对rsc1285A1x和rsc1285B2H对所得菌落进行菌落PCR验证,其中DNA片段为1.2 kb的菌落为rsc1285缺失菌株(野生型所得DNA片段为2.2 kb)。

利用该方法将pK18d1285分别融合到青枯菌RK5050(OE1-1 popA-lacZAY)[17]、RK5046(OE1-1 hrpB-lacZYA)、RK5120(OE1-1 hrpG-lacZYA)、5212(OE1-1 prhG-lacZYA)中,最终得到rsc1285基因缺失菌株RK5701(popA-lacZAY,Δrsc1285)、RK5704(hrpB-lacZAY,Δrsc1285)、RK5707(hrpG-lacZAY,Δrsc1285)和5710(prhG-lacZAY,Δrsc1285)。

图 1 青枯菌rsc1285基因缺失突变体的构建 Figure. 1 Construction of rsc1285 mutant with in-frame deletion. A: Schematic diagram of gene in-frame deletion using pK18mobSacB based homologous recombination (modified from[16] ); B: Confirmation of rsc1285 mutant via PCR.M: 5000 bp DNA ladder (TaKaRa); lane 1: rsc1285 deletion mutant RK5701; lane 2: wild type strain RK5050.
1.3 互补菌株的构建

本实验利用Tn7T(pUC18mini-Tn7T-Gm)介导的定点插入体系进行互补研究,目标DNA经该系统可定点插入到青枯菌glms基因下游25 bp处(图2)[18]。以青枯菌OE1-1基因组DNA为模板,rsc1285A1x和rsc1285B3H(5′-CGAAGCTTCT ATGGATGTTCGTTGCCGGTA-3′)为引物扩增rsc1285基因(含上游约600 bp区域,可能包含其启动子),所得PCR产物(1.6 kb)经琼脂糖凝胶电泳纯化、连接到EcoR V 酶切的pBluescript II KS(+)质粒上。所得质粒经酶切、测序验证后,命名为pKS1285C,并用于下一步互补质粒构建。用BamH I-HindIII双酶切pKS1285C,琼脂糖凝胶电泳分离,提取1.6 kb片段并连接到BamH I-HindIII双酶切的pUC18mini-Tn7T-Gm中。所得质粒经酶切、测序验证合格后,用于互补实验,命名为pUC1285。pUC1285和含转座酶的质粒pTNS2经电击,共同导入到RK5701菌株中,并进行Gm抗性筛选,对所得菌落用引物对glmsdown(5′-GCGCTCAAGCTCAAGGAGATC-3′)[7]和Tn7R(5′-CACAGCATAACTGGACTGATTTC-3′)[18]进行菌落PCR验证后用于互补实验(目标片段为500 bp),所得菌株命名为RK5713 (popA-lacZYA Δrsc1285/rsc1285)。

图 2 基于Tn7T定点插入体系的互补菌株构建 Figure. 2 Construction of complemented strains using Tn7T based site-specific integration system. A: Structure of pUC18-mini-Tn7T-Gm (modified from [18]); B: Schematic diagram of site-specific integration into the chromosome of R. solanacearum; C: Confirmation of rsc1285 complemented strain via PCR. M: 10 kb DNA ladder (TaKaRa); lane 1, 2: two complemented strains (including RK5713).
1.4 β-半乳糖苷酶活性测定

本实验以报告基因lacZYA的表达水平来表征融合基因的表达水平,lacZ编码β-半乳糖苷酶活性的测定参照Yoshimochi等[17]方法。青枯菌在hrp 诱导培养液中培养一定时间后(OD600约为0.1)用于酶活测定,每个实验至少重复3次后计算平均值和标准方差。

1.5 致病性测定

参照Zhang和Yao等[8, 19]接种法,采用灌根接种和叶柄伤口接种两种方法侵染番茄易感品种(Solanum lycopersicum cv. Moneymaker)。灌根接种时按107 CFU/g土壤的标准进行,叶柄伤口接种时,将2 μL的107 CFU/mL菌液滴到新切的叶柄伤口处。每菌至少侵染12株番茄植株,每天观察发病情况,持续观察2-3周,参照Meng等 [20] 调查方法按0-4级记录病情指数。每个实验至少重复3次后计算平均值和标准方差。

1.6 青枯菌在番茄植株内生长测定

参照Zhang等[8]方法测定青枯菌在番茄植株内的生长情况。青枯菌以叶柄伤口接种番茄植株后,分别在接种4 d和7 d后(分别对应病情指数1和4),切取地上5 cm处番茄茎部(约1 cm长),称重、研磨后稀释铺平板、计算菌数。每个实验至少重复3次后计算平均值和标准方差。

2 结果和分析 2.1 青枯菌rsc1285基因缺失突变体的构建与鉴定

青枯菌Δrsc1285突变体的构建分别经过Km和蔗糖筛选,其中第二步的蔗糖筛选是利用质粒上蔗糖致死基因挑选不含质粒的菌株(即基因缺失突变体和野生型回复突变体)。对最终筛选到的菌落以rsc1285A1x和rsc1285B2H为引物进行PCR扩增,其中PCR片段大小为1.2 kb的菌落为rsc1285基因敲除突变体(野生型对应2.2 kb片段,图1-B)。从琼脂糖凝胶中提取1.2 kb的DNA片段并送交上海生工公司进行测序,选rsc1285基因缺失,但其上下游基因序列未发生碱基突变的菌落为rsc1285基因缺失突变体。通过该方法分别得到rsc1285基因敲除菌株RK5701(popA-lacZAY Δrsc1285)、RK5704(hrpB-lacZAY Δrsc1285)、RK5707(hrpG-lacZAY Δrsc1285)和5710(prhG-lacZAY Δrsc1285)。

2.2 互补菌株的构建与鉴定

1.6 kb的rsc1285基因(含上游约600 bp区域,可能包含启动子)经pUC18-mini-Tn7T -Gm介导插入到青枯菌glms基因下游25 bp处,筛选具有Gm抗性的菌落并以glmsdown和Tn7R为引物进行PCR扩增,所得片段为500 bp的菌落表示rsc1285基因的插入(图2-C),所得互补菌株命名为RK5713(popA-lacZAY Δrsc1285/rsc1285)。

2.3 Rsc1285对青枯菌popA(T3SS)表达的影响

rsc1285因转座子插入失活后,青枯菌OE1-1的popA表达水平明显降低,表明Rsc1285可能是popA(T3SS)基因表达必需的。在RK5701(popA-lacZAY Δrsc1285)中,其popA的表达水平降低为98(±19)Miller单位,明显低于野生型的341(±25)Miller 单位(图3),该结果和转座子突变体结果一致。而在互补突变体RK5713(popA-lacZAY Δrsc1285/rsc1285)中,rsc1285基因的导入可以把RK5701的popA表达水平恢复到野生型水平(图3)。这些结果证实Rsc1285是青枯菌popA(T3SS)基因表达的一个重要影响因子。

图 3 Rsc1285对青枯菌popA表达水平的影响 Figure. 3 Effect of Rsc1285 on popA expression in R. solanacearum. RK5050 :popA-lacZYA; RK5701:popA-lacZAY Δrsc1285; RK5713:popA-lacZYA Δrsc1285/rsc1285.
2.4 Rsc1285对青枯菌致病力的影响

以伤根接种法侵染番茄植株时,青枯菌RK5050接种的番茄植株在接种4 d后出现枯萎症状(病情指数为1),接种10 d后全部枯萎死亡(病情指数为4);RK5701接种的番茄植株在接种6 d后出现枯萎症状(病情指数约为0.5),接种10 d后病情指数达2.1,接种16 d后病情指数达到最高,约为3.3,两者差异显著(P<0.05或0.01),而在互补突变体RK5713中,rsc1285基因的导入可以把RK5701的弱致病力恢复到野生型水平(图4)。这些结果证实Rsc1285是青枯菌对寄主植物致病的一个重要因子。

图 4 青枯菌Rsc1285对致病力的影响 Figure. 4 Effect of Rsc1285 on pathogenicity of R. solanacearum. RK5050:popA-lacZYA;RK5701:popA-lacZAY Δrsc1285;RK5713:popA-lacZYA Δrsc1285/rsc1285. *, P<0.05;**, P<0.01.
2.5 Rsc1285与青枯菌在培养液和寄主体内生长的关系

液体培养时(B 培养液和hrp诱导培养液)中,RK5701展现出和野生型(RK5050)完全相同的生长曲线(数据未显示)。当以叶柄伤口接种法侵染番茄植株时,RK5701在番茄茎部也展现和RK5050相同的生长趋势,4 d后约为 108 CFU/g,7 d后约为1010 CFU/g(图5),表明Rsc1285不影响青枯菌在培养液和寄主体内的生长。

图 5 Rsc1285与青枯菌在寄主体内生长的关系 Figure. 5 Effect of Rsc1285 on bacterial growth in planta. RK5050:popA-lacZYA;RK5701:popA-lacZAY Δrsc1285.
2.6 Rsc1285调控青枯菌hrpB基因的表达,但和hrpGprhG表达无关

青枯菌hrp基因簇及众多T3Es基因的表达受HrpB的直接控制,而hrpB基因的表达受HrpG和PrhG的正向调控。当rsc1285基因被敲除后,hrpB-lacZYA的表达水平降低为65(±9)Miller单位,明显低于野生型的173(±11)Miller 单位(图6),表明Rsc1285是青枯菌hrpB基因表达的一个重要因子。而rsc1285基因的缺失并不影响hrpGprhG的表达水平(图6),表明Rsc1285通过HrpB控制T3SS基因的表达,但和HrpG、PrhG无关。

图 6 青枯菌Rsc1285与hrpB、hrpG和prhG表达的关系 Figure. 6 Effect of Rsc1285 on the expression of hrpB, hrpG and prhG in R. solanacearum. From left to righrt, RK5046 (hrpB-lacZAY),RK5704(hrpB-lacZAY Δrsc1285);RK5120 (hrpG-lacZAY), RK5707(hrpG-lacZAY Δrsc1285); RK5212 (prhG-lacZAY), 5710 (prhG-lacZAY Δrsc1285). Black bar, wild type; gray bar, rsc1285 deletion mutant.
3 讨论

作为世界上分布最广、危害最严重的十大植物病原细菌之一,青枯菌的致病机理一直都是大家广泛研究的热点。其中注射器状的T3SS是青枯菌与寄主细胞互作、产生致病性的一个决定因子,而HrpB直接控制hrp基因簇、T3SS及众多T3Es的表达。迄今,大量研究揭示了青枯菌调控hrp基因簇及T3SS的精密网络,但最近的一些研究表明青枯菌T3SS被调控的机制远比科学家想象的复杂和精妙[21, 22]。如Schmidtke等通过转录组研究发现一些小RNA和T3SS相关[23];Monteiro和Jacobs等从体内(in planta)互作角度重新审视青枯菌的致病过程并取得了一些全新的发现[24, 25];我们之前也通过构建转座子突变体文库也筛选到了一些影响青枯菌hrp基因簇表达的未知因子[8],因此研究青枯菌hrpB、T3SS等转录表达被调控的途径,有助于我们从分子水平进一步了解青枯菌致病的全局性调控网络。

Rsc1285是我们前期通过转座子突变技术筛选到的一个可能影响hrp基因簇及T3SS表达的正向调控因子[8]。本文通过基因敲除、基因互补等实验初步研究了Rsc1285对青枯菌hrpB、T3SS转录表达和致病性的影响。在青枯菌GMI1000基因组中,Rsc1285被注释为可能和原核细胞染色体分离及染色质凝集相关的蛋白B (Putative prokaryotic chromosome segregation and condensation protein B,ScpB),可能和细胞的分裂过程相关[26, 27]。此外,通过和Ralstonia eutropha ATCC 17699、H16、 DSM 428、337等菌株比较,Rsc1285也可能是一个转录调控因子(https://iant.toulouse.inra.fr/bacteria/annotation/cgi/ralso.cgi)。但本研究发现,无论在营养丰富型、贫瘠型培养液中,还是在寄主番茄植株体内,rsc1285缺失突变体都展现出和野生型菌株相同的生长特性,表明Rsc1285可能并不是青枯菌细胞分裂过程的重要影响因子。当rsc1285基因缺失时,青枯菌hrpB、T3SS的转录表达水平明显降低,表明Rsc1285通过HrpB影响了青枯菌T3SS的转录表达。青枯菌hrpB 的转录表达受到HrpG和PrhG的共同正向调控[8, 9],但Rsc1285的有无并不影响青枯菌hrpG和prhG的转录表达水平,因此,Rsc1285可能通过一个全新的途径调控青枯菌hrpB 的转录表达。当侵染寄主番茄植株时,rsc1285缺失突变体较野生型菌株的致病力显著减弱,其较野生型菌株发病晚2 d,且发病程度相对减弱,但致病性并未完全丧失,表明Rsc1285在一定程度上参与了青枯菌对寄主植物的致病过程,但不是致病的决定因子。此外,进一步研究表明:在运动性和生物膜形成方面,rsc1285缺失突变体较野生型无明显差异(数据未显示)。因此,Rsc1285通过一个全新的途径控制青枯菌的致病过程,该结果有助于我们从分子水平进一步了解青枯菌致病的全局性调控过程。

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青枯菌Rsc1285参与Ⅲ型分泌系统及致病力的调控
李牧原, 徐鹏霞, 张洧琪,