孙轶芳, 赵鹏, 刘元, 李友国
华中农业大学农业微生物学国家重点实验室, 湖北 武汉 430070
收稿日期：2019-12-18；修回日期：2020-03-26；网络出版日期：2020-09-15
基金项目：国家重点研发计划（2019YFA09004700，2018YFD0201006）；国家自然科学基金（31670243）；华中农业大学科技创新基金（2662017PY052，2662017PY121）
*通信作者：李友国, Tel:+86-27-87281685;Fax:+86-27-87280670;E-mail:youguoli@mail.hzau.edu.cn.
摘要：[目的] 研究Sinorhizobium fredii SMH12中的nopP在共生固氮过程中的功能，为深入解析根瘤菌效应蛋白的菌植互作机理提供线索，进而为大豆高效根瘤菌的遗传改良提供一定的科学依据。[方法] 利用生物信息学分析nopP的结构特征，构建nopP缺失、过表达和互补菌株，并对其进行共生表型分析；通过qRT-PCR分析nopP在共生过程中的时空表达特征，测定在接野生型和突变体的冀豆17中NIN、ENOD40、PR1、PR2和PR5的表达量；采用激光共聚焦显微镜观察NopP的亚细胞定位。[结果] 根瘤菌的NopP不包含任何已知功能域，与病原体的任何Avr效应物没有同源性。nopP缺失之后对冀豆17和中黄13的根瘤固氮酶活均有显著影响，在瘤数上对冀豆17有显著增加，表明nopP突变后促进其与冀豆17和中黄13的共生固氮。qRT-PCR显示，nopP在自生条件下少量表达，在共生条件下表达量显著升高，尤其在接菌2 d后表达量达到最高，显示该基因可能与根瘤菌早期侵染相关。此外，发现NopP在烟草叶片和大豆根中均定位于细胞膜和细胞核。接种突变体的冀豆17根中NIN的表达量升高1.2倍，PR5的表达量降低3.6倍。[结论] 效应蛋白NopP在与大豆共生过程中，参与根瘤菌的早期侵染以及在根瘤菌与豆科宿主植物之间的免疫防御反应中发挥重要功能。
关键词：共生固氮大豆快生根瘤菌SMH12效应蛋白NopP共生表型
Functional characterization of a T3SS effector protein NopP of Sinorhizobium fredii SMH12 in the symbiotic nitrogen fixation and interaction with soybean
Yifang Sun, Peng Zhao, Yuan Liu, Youguo Li
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, Hubei Province, China
Received: 18 December 2019; Revised: 26 March 2020; Published online: 15 September 2020
*Corresponding author: Youguo Li, Tel:+86-27-87281685;Fax:+86-27-87280670;E-mail:youguoli@mail.hzau.edu.cn.
Foundation item: Supported by the National Key Research and Development Project (2019YFA09004700, 2018YFD0201006), by the National Natural Science Foundation of China (31670243) and by the Fundamental Research Funds for Huazhong Agricultural University (2662017PY052, 2662017PY121)
Abstract: [Objective] To understand the molecular mechanism of the effector nopP in the nodulation and nitrogen fixation of Sinorhizobium fredii SMH12, and to provide foundation for effective genetic improvement of rhizobium-soybean symbiosis, we studied the function of nopP in symbiotic nitrogen fixation between S. fredii SMH12 and soybean. [Methods] The phylogenetics of the NopP was analyzed by bioinformatics, and the deletion mutant (ΔnopP), overexpression (OE-nopP) and complementation strains (CM-nopP) were constructed by genetic operation. We further analyzed the symbiotic phenotypes. Meanwhile, the expression pattern of nopP under free-living and symbiotic conditions and the expression of NIN, ENOD40, PR1, PR2 and PR5 in ΔnopP mutant in roots of Glycine max cv. JD17 inoculated with SMH12 and SMH12 ΔnopP were detected by quantitative real-time PCR (qRT-PCR). The subcellular localization of NopP was observed by laser confocal microscopy. [Results] The NopP protein of S. fredii SMH12 does not contain any functional domains and has no homologues with Avr effectors in the genome of several pathogens. Deletion of nopP in SMH12 significantly increased the nitrogen fixation activity of JD 17 and Zhong Huang 13 compared with the wild type strain SMH12, and the nodule number significantly increased in JD17 inoculated with the mutant ΔnopP. It is indicated that the deletion of nopP promoted the symbiotic nitrogen fixation with JD17 or Zhong Huang 13. qRT-PCR showed that the expression level of nopP under symbiosis was significantly up-regulated compared to that under free-living condition, and the nopP was highly expressed at a 2-day post inoculation (dpi), indicating that the nopP gene plays important role in the rhizobia early infection and the symbiotic nitrogen fixation. In addition, the NopP protein localizes at the cytoplasm membrane and nucleus in both tobacco leaves and soybean roots. Moreover, the expression of NIN in the roots of JD17 inoculated with the mutant ΔnopP was up-regulated by 1.2 times, whereas the expression level of PR5 was down-regulated by 3.6 times compared to the wild-type SMH12 inoculation. [Conclusion] The effector protein NopP of SMH12 plays important roles in the early infection of rhizobia and the defense response regulation in soybean under symbiosis.
Keywords: symbiotic nitrogen fixationSinorhizobium fredii SMH12effector protein NopPsymbiotic phenotypes
病原菌使用不同的策略来破坏植物防御促进感染，而T3SS是实现这一目标的重要武器。同样，共生过程中T3SS对于有效的结瘤和宿主范围也是重要的[1]。当宿主植物被初次感染时，根瘤菌表面保守的微生物相关分子模式(MAMPs)被特定的植物表面受体识别(PRR)，引起了局部较弱并且短暂的MAMP触发的免疫反应(MTI)，随后被多种机制抑制，包括细胞外多糖、结瘤因子等[2]。同时，根瘤菌通过T3SS分泌效应蛋白到宿主细胞中，抑制植物防御反应，为其繁殖提供有益的环境[3-4]。然而，效应蛋白也可以被特定的植物抗性(R)蛋白识别并诱导强烈的防御反应即效应蛋白触发的免疫(ETI)，进而导致局部超敏反应(HR)，以阻断侵染。但是，一些效应蛋白也能够抑制ETI反应，从而使其定殖[5-6]。
T3SS在许多根瘤菌中已鉴定出来，并且也鉴定到通过该分泌系统分泌的一些蛋白，这些蛋白统称为结瘤外蛋白(Nops)，能够影响宿主范围以及共生效率[7]。在B. japonicum中，预测超过30个基因可能是Ⅲ型效应蛋白，在S. fredii中，大约有15个基因是可能的Ⅲ型效应蛋白[8]。Okazaki等已经证明，根瘤菌Nops还可以通过劫持由结瘤因子(nod factor，NF)诱导的结瘤信号并直接激活宿主共生信号[9]。在效应蛋白中，NopL、NopC和NopP为根瘤菌特异性蛋白，其在植物和动物病原体中没有同源物[10]。在NGR234中，NopL抑制豆科植物根瘤提早衰老并且影响MAPK信号通路，作为MAP激酶的磷酸化底物抑制本氏烟中的宿主防御反应[11]。在S. fredii HH103基因组中，NopC能够促进结瘤[12]。NGR234中nopP的突变导致宿主豆科植物千斤拔(Flemingia congesta)和非洲山毛豆(Tephrosia vogelii)的结瘤数量显著减少[13]。HH103中，nopP的失活有利于G. max (L.) Merrill cv. Williams的结瘤，但是不利于对热带豆科植物Erythrina variegata的结瘤。并且发现nopP突变体接种到Williams上时，根部病程相关基因PR1的转录水平降低，表明NopP对该宿主植物的共生起负调作用[14]。B. diazoefficiens USDA122的NopP是介导Rj2-大豆共生不相容的决定因素，Rj2蛋白属于R蛋白的TIR-NBS-LRR类[15-16]。对USDA122中的nopP突变分析表明NopP中的R60、R67和H173三个残基是介导Rj2的不相容性所必需的。表明Rj2-大豆通过由Rj2蛋白介导的效应蛋白触发的免疫来监测NopP的特定变体并阻碍USDA122侵染[17]。但对NopP蛋白在快生根瘤菌中的功能却知之甚少。
Sinorhizobium fredii SMH12是一种广宿主根瘤菌，可与大豆及其他几十种豆科植物结瘤固氮，也是一种具有良好应用前景的根瘤菌接种剂[18]。因此，本研究对SMH12中的nopP进行共生表型鉴定和机制分析，旨在为深入研究大豆快生根瘤菌效应蛋白的菌植互作机制提供科学依据和实验材料。
1 材料和方法 1.1 材料
1.1.1 菌株、质粒和培养基: 所用菌株和质粒见表 1。根瘤菌(Sinorhizobium fredii HH103)采用YMA培养基培养，筛选突变体时采用AMS培养基，培养条件是28 ℃培养；大肠杆菌(Escherichia coli，E. coli)采用LB培养基，培养条件是37 ℃。
表 1. 本研究所用菌株和质粒 Table 1. Bacterial strains and plasmids used in this study
| Strains and plasmids | Characteristics or function | Sources |
| E. coli DH5α | Host of recombinant plasmids | This lab |
| E. coli S17-1 | The helper strain used for conjugation | This lab |
| GV3101 | Host of recombinant plasmids | This lab |
| Sinorhizobium fredii SMH12 | Wide type of Sinorhizobium fredii, Strr | This lab |
| SMH12 (ΔnopP) | In frame deletion of nopP from SMH12, kanr | This study |
| CM-nopP | Complement of nopP for SMH12ΔnopP, Gmr | This study |
| OE-nopP | Overexpression of nopP for SMH12, Gmr | This study |
| PMP2463 | Gene fusion expression localization vector, Gmr | This lab |
| pCM351 | Mutant strains construction vector, Gmr | This lab |
| pCM158 | Mutant strains construction vector, Kanr | This lab |
| pBBR1MCS5 | Gene fusion expression vector, Gmr | This lab |
| pCAMBIA1302 | Gene fusion expression localization vector, Kanr | This lab |
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1.1.2 植物材料: 冀豆17、中黄13、Glycine max Williams 82、BD2、圣豆10号、绥农14和天隆一号(均来自王学路实验室)。
1.1.3 主要试剂和仪器: 限制性内切酶、反转录酶、RNase inhibitor购自Fermentas公司，pMD19-T(simple)、DNA连接酶购自TaKaRa公司，PCR反应相关试剂、琼脂糖凝胶电泳Marker购自东盛公司；抗生素、培养基相关等分子生物学试剂均购自中国国药集团。本研究所用PCR引物的合成和DNA的测序均由武汉天一辉远有限公司和擎科生物科技有限公司完成；荧光定量PCR仪为ABIStep One，气相色谱仪为GC4000A。
1.1.4 引物: 本研究所用引物见表 2，引物均用Primer 5.0软件设计。
表 2. 本研究所用引物 Table 2. Primers used in this study
| Primers | Sequences of primers (5′→3′) | Brief description |
| nopP-up-F | ggtaccATCATCTGGCAATCGGTT | Amplifying the exchange upstream arm fragment |
| nopP-up-R | catatgACGAGCTATCAATTCGACC | |
| nopP-down-F | gggcccGACTTACGAAGATGACTTCATG | Amplifying the exchange downstream arm fragment |
| nopP-down-R | accggtCATCTACTTATGAGCTCCAGC | |
| nopP-Map-F | AGTCGGGACGCAATGGAT | Validation of nopP mutants |
| nopP-Map-R | ACTCCACTTCCAATCACTCCG | |
| nopP-ORF-F | ATGTACGGTCGAATTGATAGCT | Cloning the ORF of nopP used |
| nopP-ORF-R | TCACATGAAGTCATCTTCGTAAGT | for complementary plasmid |
| Gm-F | ATGTTACGCAGCAGCAACG | Cloning the ORF of Gm |
| Gm-R | TTAGGTGGCGGTACTTGGG | |
| nopP-q-F | AGGTGGGTTCAGCATGGAAG | Using for nopP of qRT-PCR |
| nopP-q-R | GCTCGAGCAGAATATCGCCT | |
| 16S-q-F | GGATCGGAGACAGGTGCTGCA | Reference gene of qRT-PCR |
| 16S-q-R | CGTGTGTAGCCCAGCCCGTA | |
| nopP-L-F | ggtaccATGTACGGTCGAATTGATAGCT | Constrution of the recomb ination of NopP localization |
| nopP-L-R | tctagaCATGAAGTCATCTTCGTAAGT | |
| PR1-F | GGCCAATACGGGGAGAATCT | Using for PR1 qRT-PCR |
| PR1-R | TCCAAACAACCTGAGTGTAATGC | |
| PR2-F | TGAGAGTGGATGGCCTTCTT | Using for PR2 qRT-PCR |
| PR2-R | TGTTTCACATTCCGAACCAA | |
| PR5-F | AACGTGCCCATGGACTTTAG | Using for PR5 qRT-PCR |
| PR5-R | CGGTTTTGAAGACAGTGCAA | |
| ENOD40-F | GGCTTCTCTGATCAACAAGGG | Using for ENOD40 qRT-PCR |
| ENOD40-R | ACATAGCCATAGAGACCCCA | |
| NIN-F | ATATGGTGGGTTGGTGCAGA | Using for NIN qRT-PCR |
| NIN-R | CCCAGCATCCTTCCACAAAC | |
| Tef-q-F | TGCAAAGGAGGCTGCTAACT | Reference gene of qRT-PCR |
| Tef-q-R | CAGCATCACCGTTCTTCAAA | |
| M13-F | GAGCGGATAACAATTTCACACAGG | Identify the recombination plasimid PMP2463-NopP |
| M13-R | CGCCAGGGTTTTCCCAGTCACGAC |
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1.2 生物信息学分析 通过NCBI网站查找目的基因序列，然后通过BLASTp比对查找同源蛋白，挑选不同种属的同源蛋白序列，利用MEGA软件构建系统发育树。
1.3 qRT-PCR检测目的基因在自生及共生下的表达量 大豆种子经氯气消毒灭菌后，放置于超净工作台吹40min，然后用无菌水浸泡种子1 h，播种于灭菌后的蛭石中，用无氮液浇灌。待幼苗长出第一片真叶时，接快生型根瘤菌SMH12，分别收取接菌后8 h、1 d、2 d、4 d、8 d、10 d、12 d、14 d、16 d、20 d、25 d的根部组织，抽提以上不同天数根部组织及自生条件下SMH12中的RNA，并反转录成cDNA，以得到的cDNA为模板、16S RNA为内参基因做荧光定量PCR。qRT-PCR反应程序如下：95 ℃ 5 min；94 ℃ 30 s，60 ℃ 20 s，72 ℃ 20 s，40个循环；72 ℃ 5 min。信号检测染料使用SYBR Green，分析相对表达量采用2–ΔΔCT值的方法[19]。
1.4 亚细胞定位分析
1.4.1 在烟草细胞中的亚细胞定位: 以SMH12基因组为模板扩增nopP编码序列并连接到GFP的N端，融合表达NopP-GFP。随后将载体转化到根癌农杆菌GV3101菌株中，过夜培养并收集菌液，使用烟草注射buffer重悬菌体(0.01 mol/L MES-OH，pH 5.8，0.01 mol/L MgCl2，0.15 mol/L乙酰丁香酮acetosyringo-ne)，使其终浓度OD600为0.5，然后室温静置4 h后用1 mL注射器从烟草叶片下表皮注射进整片烟草叶片，培养2 d后使用激光扫描共聚焦显微镜(Zeiss LSM510)观察细胞荧光并拍照。
1.4.2 在共生状态下的亚细胞定位: 大豆种子经氯气消毒灭菌后，放置于超净工作台吹40 min，用无菌水浸泡种子1 h，播种于灭菌后的蛭石中，用无氮液浇灌。待幼苗长出第一片真叶时，接快生型根瘤菌SMH12，分别收取接菌后2 d和8 d的植物根部组织，使用激光扫描共聚焦显微镜(Zeiss LSM510)观察细胞荧光并拍照。
1.5 nopP缺失、超表达及互补菌株的构建
1.5.1 ΔnopP突变体的筛选与鉴定: 利用Crelox系统双交换置换缺失的方法[20]，构建nopP缺失突变体。以SMH12基因组DNA为模板，采用nopP-up-F/R和nopP-down-F/R引物对，分别扩增nopP同源交换上臂和下臂，将产物连接到载体
pMD19-T (simple)，测序正确后将其双酶切后分两步与消化的pCM351载体连接，得到质粒pCM351::nopP-up-down转化至E. coli S17-1中，经两亲本结合转移，稀释涂布于SM+Str+Gm平板上，经引物对nopP-MAP-F/R及Gm-F/R筛选转化子，进行PCR验证突变体。
1.5.2 超表达和nopP互补菌株的构建: 以快生型根瘤菌SMH12基因组DNA为模板，用nopP-ORF-F/R引物对，扩增得到nopP完整的开放阅读框，经酶切回收后与同样双酶切的载体pBBR1MCS-5连接，构建重组表达载体pBBR5-nopP，测序正确后进行两亲本接合转移，分别将重组载体导入野生型SMH12及突变体ΔnopP中，稀释涂布于含有相应抗生素的平板上，倒置于28 ℃培养箱中，培养4–7 d后，以引物对M13-F/R进行阳性转化子PCR验证。
1.6 根瘤菌-大豆共生表型分析 大豆种子经氯气消毒灭菌后，放置于超净工作台吹40 min，用无菌水浸泡种子1 h，播种于灭菌后的蛭石中，用无氮液浇灌。待幼苗长出第一片真叶时，分别接野生型SMH12(WT)、突变体菌株ΔnopP、超表达菌株nopP(OE)、互补菌株(CM)，收取接菌后25 d植物，分别统计野生型和突变体植物的地上部分鲜重、根瘤数量、根瘤重量及固氮酶活。
1.7 大豆共生及免疫防御相关基因的表达量分析 分别收取接菌野生型SMH12和突变体ΔnopP后2 d的冀豆17根部组织，抽提以上不同根部组织的RNA，并反转录成cDNA，以得到的cDNA为模板，TEF为内参基因做荧光定量PCR，分析野生型和突变体中NIN、ENOD40、PR1、PR2和PR5的表达量。qRT-PCR反应程序如下：95 ℃ 5 min；94 ℃ 30 s，60 ℃ 20 s，72 ℃ 20 s，40个循环；72 ℃ 5 min。信号检测染料使用SYBR Green，相对表达量分析采用2–ΔΔCT值的方法。
2 结果和分析 2.1 NopP的系统发育进化树 前期，我们对Sinorhizobium fredii SMH12基因组进行了测序和注释，并且通过同源比对发现S. fredii SMH12基因组中含有一个可能的nopP，进一步通过BLASTp (https://blast.ncbi.nlm.nih.gov/Blast.cgi)分析发现，在快生型、慢生型和中慢生型根瘤菌中均存在NopP同源蛋白。系统发育树分析显示，NopP的同源蛋白分为3个簇，分别为相似度较高的慢生根瘤菌属(Bradyrhizobium)、快生型的中华根瘤菌属(Sinorhizobium)和中慢生根瘤菌属(Mesorhizobium) (图 1)。氨基酸比对分析发现，NopP在同一菌属中相似度较高，相似度均超过90%，而在不同菌属中相似度则较低，不超过40%。另外，我们在其他非根瘤菌属中未检索到NopP同源蛋白的存在，表明NopP是根瘤菌中所特有的效应蛋白，且保守性较高，可能是在共生的根瘤菌系统中进化来的。
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| 图 1 NopP同源蛋白的系统发育进化树 Figure 1 Neighbour-joining phylogenetic tree of NopP homologues. Phylogenetic tree of NopP and its homologs from other species was constructed by the neighbor-joining method using the MEGA version X program. Numbers in bracket represent the sequences accession number in GenBank. The number at each branch points is the percentage supported by bootstrap. Bar, 0.05 sequence divergence. |
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2.2 根瘤发育过程中nopP的表达特征 为了明确nopP在根瘤发育过程中的表达特征，我们采用qRT-PCR方法分别对接菌处理的大豆根瘤样品进行了表达量分析。结果显示，SMH12中的nopP在自生和共生过程中均可表达。其中在接菌2 d后，nopP表达量最高，是自生条件的52倍，随后表达量逐渐下降，并趋于稳定，但相对于自生条件仍然高量表达(图 2)，表明nopP在共生过程中发挥重要作用。
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| 图 2 nopP在自生及共生不同时期的动态表达 Figure 2 The dynamic expression patterns of nopP under free-living condition and different staged root nodules. From left to right: SMH12 of free-living cells; RNA was isolated from the roots at 8 h, 1, 2, 4, 8, 10, 12, 14, 16, 20 and 25 days after inoculation respectively. The primers listed in Table 2 as described in Methods. The expression level of each gene was normalized by the 16S rRNA gene. Data presented are means±standard deviations of three independent experiments. |
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2.3 NopP蛋白的亚细胞定位 为了探明NopP在细胞水平下的定位，我们对生长约4周的烟草进行了农杆菌瞬时表达转化，并在注射48 h后观察NopP的定位。结果显示，在烟草叶片细胞表面和细胞核中均能观察到绿色荧光，并进一步通过DAPI染色，观察到圆点状的蓝色荧光与绿色点状荧光重合，明确NopP定位于细胞核中(图 3-A)，表明NopP在烟草中定位于细胞膜和细胞核中。
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| 图 3 NopP在烟草和共生状态下的亚细胞定位 Figure 3 Expression of NopP::GFP in tobacco epidermal (A) and soybean root cells (B). N. benthamiana leaves and soybean roots were transfected with combinations of constructs for the expression of NopP::GFP. Confocal images of tobacco cells and soybean root cells expressing NopP fused to green ?uorescent protein (GFP). Tobacco leaves were taken 24–48 h after transfection. Soybean root tissues were collected at 2 and 8 days after inoculation. Scale bars, 25 μm (A) and 50 μm (B). |
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为了进一步明确NopP的亚细胞定位，我们将SMH12(pMP2463-nopP)接种于冀豆17中，并分别取接菌2d和8d后的植物根部，随后将根部进行琼脂糖包埋做成切片，再用激光共聚焦观察。结果显示：NopP在细胞膜和细胞核中均能观察到荧光(图 3-B)，与在烟草叶片中观察的结果相一致。因此，明确NopP定位于细胞膜和细胞核中。
2.4 ΔnopP突变菌株的构建和鉴定 以SMH12总基因组DNA为模板，分别扩增大小为692 bp和691 bp的同源上臂和下臂(图 4-A)，同源上臂经Kpn I和Nde I连到载体pCM351，再经Apa I和Age I将同源下臂连到载体pCM351::up。将构建好的重组质粒导入快生型根瘤菌SMH12中，重组质粒上的交换片段与根瘤菌基因组中的nopP上、下游片段发生同源重组，整合到染色体上，进而将该基因置换。以载体pCM351的引物Gm-F与酶切位点对应的目的基因的筛选引物nopP-MAP-R进行PCR验证扩增出约为1625 bp (图 4-B)片段，大小与预期相符。同时采用野生型SMH12总DNA模板为阴性对照，无条带扩出。进一步将pCM158导入SMH12 (?nopP::Gm)，将Gm消除得到完全缺失突变体?nopP，并进行PCR验证，结果表明SMH12 (?nopP)突变体构建正确(图 4-C)。
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| 图 4 ?nopP突变株的PCR鉴定 Figure 4 Identification of SMH12 (?nopP) mutant by PCR. M: DL2000 DNA marker. A: PCR amplification of exchanging arms. Lane 1: the exchange upstream arm; lane 2: the exchange downstream arm. B: PCR verificate of SMH12::Gm. Lane 1: the post-gene exchange vector fragment; lane 2: the SMH12 wild-type genome. C: From 1 to 3: cloning the ORF of nopP validates the wild type SMH12 genome, SMH12 (ΔnopP), and the elimination the Gm resistant mutant. |
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2.5 ΔnopP突变体与冀豆17和中黄13的共生固氮表型分析 为了研究SMH12 (?nopP)对共生固氮的影响，也构建了nopP超表达OE-nopP及互补CM-nopP，并考察各菌株与冀豆17和中黄13的共生表型。结果表明，冀豆17和中黄13接种不同菌株地上部分长势均有差异，冀豆17接种突变体后的长势高且叶片为绿色，接种超表达的与野生型相比长势较弱且发黄。接种互补菌株的植物表型介于野生型与突变体之间，可部分恢复到野生型表型(图 5-A)。同样，中黄13接种突变体和接种超表达的地上植物长势均较野生型的高且叶片为绿色，接种互补菌株的植物表型介于野生型与突变体之间，可部分恢复到野生型表型(图 5-B)。
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| 图 5 ?nopP突变株与冀豆17和中黄13的共生表型 Figure 5 Symbiotic phenotype induced by strains tested on JD17 and ZH13. A: Symbiotic phenotype induced by strains tested on JD17; B: Symbiotic phenotype induced by strains tested on ZH13. WT: the wild type SMH12; ?nopP: the nopP mutant strain; CM: the nopP complem-entation strain; OE: the nopP overexpression strain. |
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进一步对植株根瘤固氮酶活、瘤数、瘤重及地上部分鲜重进行统计分析，结果表明，冀豆17接种突变体植株的固氮酶活为24.3 μmol/(g·h)，野生型植株的共生固氮酶活为11.1 μmol/(g·h)，突变株的固氮酶活相较于野生型显著升高(P < 0.05)，瘤数也显著高于野生型。接种超表达菌株的植物根瘤酶活、瘤数、瘤重及地上部分鲜重均显著低于野生型。接种突变体的中黄13植株的根瘤固氮酶活较野生型显著升高，接种超表达菌株的植株根瘤固氮酶活为8.6 μmol/(g·h)，野生型植株的为23.3 μmol/(g·h)，超表达的固氮酶活相较于野生型显著降低(P < 0.01)。接种突变体和超表达的瘤数、瘤重以及地上部分鲜重相较于于野生型无明显差异(图 6)。总之，nopP突变后影响了根瘤的生长发育，导致冀豆17和中黄13的根瘤固氮酶活性升高，从而促进了植物的生长发育，表明nopP的功能与共生固氮作用相关。
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| 图 6 植株根瘤固氮酶活、瘤数、瘤重及地上部分鲜重的比较测定 Figure 6 Comparative quantification of the nitrogenase activity (A), number of nodules (B), weight of nodules (C) and fresh weight (D) of aerial part induced by strains tested on JD17 and ZH13. WT: the wild type SMH12; ?nopP: the nopP mutant strain; CM: the nopP complementation strain; OE: the nopP overexpression strain. *: significant difference at P < 0.05; **: significant difference at P < 0.01; the error bars represent the standard deviations of three independent experiments. |
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2.6 冀豆17根中共生和免疫相关基因的表达量分析 通过接种突变体菌与野生型菌相比，在接菌2 d后的冀豆17根中NIN (在NFR感知NFs后表征良好的共生基因)的表达量升高1.2倍，ENOD40表达水平没有显著变化。为了检查在冀豆17根中nopP是否激活了防御反应，我们监测了冀豆17根中防御标记基因(PR1，PR2和PR5)的表达。结果表明，与接种野生型的相比，接种突变体菌的冀豆17根中PR5在接菌2 d后的表达量显著降低3.6倍。相反，PR1和PR2的表达没有显著改变(图 7)。
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| 图 7 NIN、ENOD40、PR1、PR2和PR5在冀豆17中的表达量 Figure 7 Expression of NIN, ENOD40, PR1, PR2 and PR5 genes in roots of JD17. G. max cv. JD17 inoculated with SMH12 and SMH12ΔnopP was determined by quantitative reverse transcription PCR using primers listed in Table 2 as described in Methods. RNA was isolated from the roots at 2 day after inoculation (DAI). The expression level of each gene was normalized by the TEF gene. Data presented are means±standard deviations of three indepen dent experiments. |
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这表明PR5的瞬时和早期表达是由野生菌引起的，可能和nopP有关。这些结果表明在与冀豆17的共生过程中，nopP可能被宿主识别后引起了防御反应，从而抑制了早期根瘤菌的侵染。
3 讨论 本室对大豆快生型根瘤菌SMH12基因组进行了测序和注释，通过生物信息分析发现其基因组中含有1个可能的nopP。前人报道，B. diazoefficiens USDA122中的NopP是介导Rj2-大豆共生不相容性的决定因素。对USDA122中nopP与USDA110的nopP分析发现只有3个氨基酸残基(R60、R67和H173)有差异，而USDA110能与Rj2-大豆正常结瘤[15]。这暗示了虽然通过系统发育分析该基因在快生型根瘤菌属中相似性较高(图 1)，但是可能发挥不同的功能。在NGR234中，nopP的突变导致宿主豆科植物千斤拔(Flemingia congesta)和非洲山毛豆(Tephrosia vogelii)的结瘤数量显著减少[13]；在HH103中，nopP的失活有利于G. max (L.) Merrill cv. Williams的结瘤，但是不利于对热带豆科植物Erythrina variegata的结瘤[14]；在USDA257中，nopP以依赖于T3SS的方式分泌到Vigna unguiculata的根瘤中[21]；USDA122的NopP是介导Rj2-大豆共生不相容性的决定因素[15]。而本研究中，SMH12中的nopP缺失后，对冀豆17和中黄13的根瘤固氮酶活均有显著影响，在瘤数上对冀豆17有显著增加(图 6)，表明nopP突变后促进了冀豆17和中黄13的共生固氮。已有研究发现T3SS的基因在早期侵染阶段以及在不同宿主植物的成熟根瘤中表达水平较高[22-23]。然而，在大豆中，NopX只在侵染线中检测到，而在成熟根瘤中没有检测到[24]。此外，蛋白质组学和转录组学方法已经揭示T3SS相关基因在大豆成熟根瘤中下调[25-26]。在接种HH103的T3SS缺失突变体后4 d，PR1的表达在根中开始增加，而在识别可能的诱导子和PR1蛋白的合成之间必定存在一定的时间间隔，表明HH103的结瘤外蛋白(Nops)在与大豆共生的早期侵染阶段中被识别，可能抑制大豆防御反应，从而促进侵染[14]。本研究通过qRT-PCR检测nopP的表达，结果显示nopP在自生条件下仅少量表达，而在共生条件下表达量显著性升高(图 3)，尤其是在接菌2 d后表达量达到最高，预示着该基因可能参与共生固氮过程并与根瘤菌早期侵染相关。此外，利用基因-报告基因(NopP-GFP)融合的方法，发现nopP基因在烟草和大豆根均定位于细胞膜和细胞核中。由此，我们分析认为NopP蛋白可能通过直接或间接调控共生以及免疫相关的基因，影响其共生固氮与根瘤菌早期侵染过程。
已发现根瘤菌T3SS(rhc)基因和宿主植物R基因负责诱导共生不相容性[27-29]。这些发现暗示宿主植物通过感知特定的根瘤菌T3SS效应蛋白，并通过防御反应抑制根瘤菌侵染[30-32]。一般而言，ETI引起快速合成水杨酸(SA)，SA响应Avr效应物必需信号分子的快速合成，随后诱导SA依赖性防御信号(例如PR1、PR2和PR5)[33-34]。在Rj4-大豆植物与Bradyrhizobium elkanii USDA61的不相容性中，SA积累并且PR1在根中的表达增加[35]。最近报道在接种USDA122后，在侵染的早期阶段瞬时诱导Rj2-大豆中的PR2基因(2 DAI)。PR2诱导的时间与G. max (L.) Merr. cv. Hardee (Rj2)根毛中侵染线(IT)发育的抑制相关。表明Rj2-大豆在结瘤的早期阶段通过接种USDA122激活宿主防御反应，并且下游防御信号传导途径可能与Rj4-共生不相容性不同[15]。将S. fredii HH103的nopP突变体接种到大豆Williams上时，根部防御基因PR1的转录水平降低，表明NopP对与该宿主植物的共生起负调控作用[14]。因此，我们进一步通过定量分析共生以及免疫防御相关基因的表达量的变化情况，发现与接种野生型菌相比，接种突变体菌的冀豆17根中NIN的表达量升高1.2倍，PR5的表达量降低3.6倍。说明NopP在进入宿主细胞过程中，NIN和PR5可能参与了这一过程，影响宿主免疫防御及根瘤菌的侵染。
本文首次研究了大豆快生型根瘤菌SMH12中的效应蛋白NopP对共生固氮的影响。我们通过生物信息学及实验结果分析，认为快生型根瘤菌SMH12中nopP基因可能在根瘤菌早期侵染及宿主的免疫防御过程中发挥重要功能，但具体的功能机制还有待进一步实验研究。因此，本文的研究结果为深入了解在共生结瘤早期根瘤菌-豆科植物相互识别的机制提供了新线索。
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