Transient Expression Assay in NahG Arabidopsis Plants Using Agrobacterium tumefaciens
使用根癌农杆菌在NahG拟南芥植株中进行瞬时表达分析   

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Araceli Castillo Garriga Araceli Castillo Garriga
Shunping Yan Shunping Yan
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本实验方案简略版
Molecular Plant
Feb 2017

 

Abstract

Agrobacterium-mediated transient expression has greatly contributed to research in molecular plant biology but has low efficiency and inconsistency in Arabidopsis thaliana (Arabidopsis). Here, we describe a simple, efficient and fast protocol to make transient gene expression in NahG Arabidopsis plants using Agrobacterium tumefaciens. This protocol has been successfully used to assess protein sub-cellular localization and accumulation, enzyme activity, and protein-protein interaction. In addition, this assay overcomes the use of Nicotiana benthamiana plants as a surrogate system for transient gene expression assays. Finally, the use of this protocol does not require complex inoculation methods or specific growth conditions, and can be used with different Agrobacterium strains with similar results.

Keywords: NahG Arabidopsis (NahG拟南芥), Agrobacterium (农杆菌), Transient expression (瞬时表达), Transient transformation (瞬时转化), Protein accumulation (蛋白质积累)

Background

Agrobacterium tumefaciens (hereafter referred to as Agrobacterium)-mediated transient transformation assays have greatly contributed to research in molecular plant biology. These methods have many advantages over the laborious and time-consuming stable transformation approaches including, among others, a higher efficiency, simplicity, and fast, consistent results when the transient transformation assays are carried out in Nicotiana benthamiana. On the other hand, these assays are inefficient and lack robustness when carried out in the model plant Arabidopsis thaliana (hereafter referred to as Arabidopsis), forcing Arabidopsis researchers to use N. benthamiana as a heterologous system, which entails obvious limitations and might generate misleading results.

Many efforts have been made in the past to increase the efficacy of Agrobacterium-mediated transient transformation in Arabidopsis (reviewed in Krenek et al., 2015). Recently, we described a protocol to perform transient gene expression using NahG Arabidopsis plants, overcoming previous limitations (Rosas-Díaz et al., 2017). Using Arabidopsis NahG plants, which contain low levels of salicylic acid (SA) due to the expression of an SA hydroxylase from the bacterium Pseudomonas putida (Lawton et al., 1995), we have been able to obtain high accumulation of marker proteins such as GUS and GFP, and carry out sub-cellular localization and protein-protein interaction experiments. Remarkably, this protocol for transient expression can be used with, at least, three widely used Agrobacterium strains, LBA4404, GV3101 and C58C1. In summary, this protocol shows that expression of the NahG transgene greatly enhances the efficiency of Agrobacterium-mediated transformation in rosette leaves in Arabidopsis, enabling the routine use of this technique in the model plant.

Materials and Reagents

  1. Materials
    1. Soil mix or substrate such as Compo Sana® Universal Ligera (COMPO, TSUSTPROF25L)
    2. Plant pots such as Desch vol 11 (Desch Plantpak, catalog number: 1055278 )
    3. Seed tray–40 cavities
    4. Plant trays
    5. Cling film
    6. Syringes 1 ml or 2 ml
    7. Tissue paper
    8. Petri dishes
    9. Falcon tubes
    10. Eppendorf tubes
    11. Pipette tips
    12. Sterile toothpicks
    13. Syringe filter 0.22 µm

  2. Biological materials
    1. Arabidopsis thaliana NahG seeds (Lawton et al., 1995)
    2. Agrobacterium tumefaciens strain (LBA4404, GV3101 or C58C1) carrying a binary vector with the gene of interest

  3. Reagents
    1. Sterile deionized water
    2. Glycerol (CARLO ERBA Reagents, catalog number: 453752 )
    3. NaCl (AppliChem, catalog number: 121659.1210 )
    4. Tryptone (Biolife, catalog number: 412290 )
    5. Yeast Extract (AppliChem, catalog number: 403687.1210 )
    6. Bacteriological agar (MICROKIT, catalog number: BCB006+ )
    7. MES (2-(N-morpholino) ethanesulfonic acid) (Sigma-Aldrich, catalog number: M2933 )
    8. MgCl2 (AppliChem, catalog number: 131396.1210 )
    9. DMSO (Sigma-Aldrich, catalog number: M81802 )
    10. Acetosyringone (Sigma-Aldrich, catalog number: D134406 )
    11. Rifampicin (Duchefa Biochemie, catalog number: R0146.0005 )
    12. Tetracycline (Sigma-Aldrich, catalog number: T3383 )
    13. Gentamycin (Sigma-Aldrich, catalog number: G3632 )
    14. Spectinomycin (Duchefa Biochemie, catalog number: S0188.0005 )
    15. Kanamycin (Sigma-Aldrich, catalog number: K4378 )
    16. LB medium (see Recipes)
    17. 1 M MES (see Recipes)
    18. 1 M MgCl2 (see Recipes)
    19. 0.1 M Acetosyringone (see Recipes)
    20. Infiltration solution (see Recipes)
    21. Antibiotics solution (depending on construct and Agrobacterium strain, see Recipes)

Equipment

  1. Sterile Erlenmeyer flasks
  2. Plant growth chamber capable of sustaining 21 °C under short-day conditions (8 h light/16 h dark) with 140-150 µmol m-2 sec-1 light intensity (Radiber SA, catalog number: AGP-1400 )
  3. Spectrophotometer capable of OD600 measurements such as Shimadzu UV-1601 (Shimadzu, catalog number: 206-67001-34 )
  4. Automatic P1000, P200 and P20 micropipettes
  5. Incubator set at 28 °C such as Incubator D-6450 Hanau (Heraeus Instruments, model: D-6450 )
  6. Incubator shaker capable of sustaining 28 °C and 180 rpm such as New BrunswickTM I26 (Eppendorf, New Brunswick scientific, model: I26, catalog number: M1324-0000 )
  7. Centrifuge for 50 ml tubes such as Rotofix 32A (Hettich, catalog number: 1206-01 )
  8. Autoclave

Procedure

  1. Plant growth conditions
    Note: The preparation of Arabidopsis NahG plants is a key step in obtaining a satisfactory transient gene expression/protein expression. In this protocol, ~4 weeks old Arabidopsis NahG plants are used for Agrobacterium infiltration.
    1. Sow seeds in water-soaked soil mix in a plant pot. Cover the pot with cling film and place it in a growth chamber with 8 h light/16 h dark cycle at 21 °C.
    2. Grow until the seedlings have two true leaves (around 7-10 days).
    3. Carefully transplant seedlings to the final destination in seed trays with 40 cavities.
    4. Grow plants for approximately 3 more weeks inside the growth chamber with 8 h light/16 h dark cycle at 21 °C.
    5. Plants are ready for infiltration when they have a robust rosette of 10-12 leaves and have not started flowering.

  2. Agrobacterium cultures
    Note: This protocol can be used with, at least, three different commonly used strains of Agrobacterium: LBA4404, GV3101 and C58C1.
    1. Using a glycerol stock and a sterile toothpick, streak the Agrobacterium clone(s) to be used in LB solid plates supplemented with the appropriate antibiotics. Place the plates inside a 28 °C incubator for 48 h to obtain fresh and single colonies.
    2. The day before starting the infiltration, start liquid Agrobacterium cultures in LB liquid medium using the fresh colonies on the plates. Pick Agrobacterium biomass from a single colony, using a sterile toothpick, place it inside a sterile Erlenmeyer flask with 20 ml LB liquid media supplemented with the appropriate antibiotics, and culture them at 28 °C and 180 rpm overnight.

  3. Infiltration
    1. Agrobacterium preparation
      1. Pour saturated cultures into 50 ml Falcon tubes. Spin down cells at 4,000 x g for 10 min.
      2. Discard LB medium supernatant by decanting. Eliminate as much supernatant as possible.
      3. Resuspend with vortex the cell pellets using 1 volume of freshly prepared infiltration buffer.
      4. After resuspension, leave cultures for 2-4 h in darkness at room temperature.
      5. Prepare a 1/20 dilution of the saturated culture, measure OD600 and calculate necessary volume to have a final OD600 of 0.05. Dilute using infiltration buffer.
    2. Infiltration procedure
      1. Fill a 1 or 2 ml needleless syringe with the resuspended culture at a final OD600 of 0.05. Perform the infiltration by pressing the syringe (without needle) on the abaxial side of the leaf while exerting counter-pressure with a fingertip on the adaxial side. Observe how the liquid spreads within the leaf if the infiltration is successful. Infiltrate whole leaves (ca. 100 µl of bacterial suspension/leave).
      2. Dry the excess of culture from the leaf surface using tissue paper (Figure 1, Video 1).
      3. Two to four days after infiltration, observe fluorescence of infiltrated proteins or harvest infiltrated leaves to do a protein extraction.


        Figure 1. Experimental procedure of transient expression in NahG Arabidopsis thaliana plants using Agrobacterium tumefaciens. A. Eppendorf tube with Agrobacterium carrying the desired construct, 1 ml syringe, 4-week-old NahG Arabidopsis plants, and piece of paper. B. Plant infiltration (see details in the procedure section).

        Video 1. Experimental procedure of transient expression in NahG Arabidopsis thaliana plants using Agrobacterium tumefaciens

Notes

  1. Some leaves might suffer necrosis after infiltration with the bacterial suspension.
  2. Growing NahG Arabidopsis under long day condition (16 h light/8 h dark cycle), might not affect the transient expression if the plants have not started flowering. However, plant leaves are bigger and easier to infiltrate when plants are grown in short day conditions.
  3. It is also possible to adjust the OD600 first and then incubate the cultures for 2-4 h in darkness.
  4. Infiltrate no more than 4-5 leaves per plant. Survival rate of the plants diminishes considerably when more leaves are infiltrated.
  5. Using this protocol the level of protein accumulation is optimal at 3 days after infiltration.
  6. Practically any leaf of the rosette can be infiltrated with positive results but the eldest leaves tend to show a lower signal and the youngest leaves tend to show a stronger response to the bacterial inoculation, with the concomitant necrosis.
  7. Drying the excess of culture is not determinant for the success of the infiltration but highly recommendable due to leaves sticking together when wet, causing higher levels of necrosis.

Recipes

  1. LB medium (1 L)
    NaCl 5 g
    Tryptone 10 g
    Yeast extract 5 g
    (Only for solid medium) Bacteriological agar 16 g
  2. 1 M MES (100 ml)
    17.5 g MES
    Complete with sterile deionized water
    Adjust the pH to 5.6
    Sterilize by filtration
    Store at room temperature
  3. 1 M MgCl2 (100 ml)
    20.3 g MgCl2
    Complete with sterile deionized water
    Sterilize by autoclaving
    Store at room temperature
  4. 0.1 M Acetosyringone (10 ml)
    0.196 g acetosyringone
    10 ml DMSO
    Prepare 1 ml aliquots and store them at -20 °C
  5. Infiltration solution (100 ml)

    Top up with sterile deionized water
    Note: Prepare infiltration solution just before use.
  6. Antibiotics solution

Acknowledgments

The authors thank Huang Tan for his technical help during the imaging. This work was supported by the Spanish Ministerio de Ciencia y Tecnología (AGL2016-75819-C2) and the Shanghai Center for Plant Stress Biology, Chinese Academy of Sciences. This protocol is derived from a previous publication (Rosas-Díaz et al., 2017). The authors declare that they have no conflict of interest.

References

  1. Krenek, P., Samajova, O., Luptovciak, I., Doskocilova, A., Komis, G. and Samaj, J. (2015). Transient plant transformation mediated by Agrobacterium tumefaciens: Principles, methods and applications. Biotechnol Adv 33(6 Pt 2): 1024-1042.
  2. Lawton, K., Weymann, K., Friedrich, L., Vernooij, B., Uknes, S. and Ryals, J. (1995). Systemic acquired resistance in Arabidopsis requires salicylic acid but not ethylene. Mol Plant Microbe Interact 8(6): 863-870.
  3. Rosas-Díaz, T., Cana-Quijada, P., Amorim-Silva, V., Botella, M. A., Lozano-Duran, R. and Bejarano, E. R. (2017). Arabidopsis NahG plants as a suitable and efficient system for transient expression using Agrobacterium tumefaciens. Mol Plant 10(2): 353-356.

简介

农杆菌介导的瞬时表达大大促进了分子植物生物学的研究,但是在拟南芥(Arabidopsis thaliana)( Arabidopsis )中效率低且不一致。 在这里,我们描述了一种简单,高效和快速的方法,用于根据根癌农杆菌在 NahG 拟南芥植物中进行瞬时基因表达。 该方案已成功用于评估蛋白质亚细胞定位和积累,酶活性和蛋白质 - 蛋白质相互作用。 此外,该测定法克服了使用本氏烟草植物作为瞬时基因表达测定的替代系统。 最后,该方案的使用不需要复杂的接种方法或特定的生长条件,并且可以与具有相似结果的不同农杆菌菌株一起使用。

【背景】根癌土壤杆菌(以下简称土壤杆菌)介导的瞬时转化试验已经对分子植物生物学研究作出了重大贡献。这些方法与费时费力的稳定转化方法相比具有许多优点,其中包括在烟草本氏烟中进行瞬时转化测定时更高的效率,简单性和快速,一致的结果。另一方面,当在模式植物拟南芥(以下称为拟南芥)中进行这些测定时,这些测定是无效的并且缺乏稳健性,从而迫使拟南芥研究人员使用 N。本氏烟草作为一种异源系统,这会带来明显的局限性,并可能产生令人误解的结果。

过去已做出许多努力来提高土壤杆菌介导的拟南芥中的瞬时转化的功效(在Krenek等人的综述中,2015年)。 )。最近,我们描述了使用NahG拟南芥植物进行瞬时基因表达的方案,克服了以前的限制(Rosas-Díaz等人,2017)。使用拟南芥属NahG植物由于表达来自细菌恶臭假单胞菌的SA羟化酶而含有低水平的水杨酸(SA)(Lawton等人, ,1995),我们已经能够获得高积累的标记蛋白如GUS和GFP,并进行亚细胞定位和蛋白质 - 蛋白质相互作用实验。值得注意的是,该瞬时表达方案可以与至少三种广泛使用的农杆菌菌株LBA4404,GV3101和C58C1一起使用。总之,该协议显示,转基因NahG的表达极大地提高了土壤杆菌介导的转化在拟南芥叶片中的转化效率,在模型工厂中常规使用这种技术。

关键字NahG拟南芥, 农杆菌, 瞬时表达, 瞬时转化, 蛋白质积累

材料和试剂

  1. 物料
    1. 土壤混合物或基质如Compo Sana Universal Ligera(COMPO,TSUSTPROF25L)
    2. 植物盆如Desch vol 11(Desch Plantpak,目录号:1055278)
    3. 种子托盘40腔
    4. 植物托盘
    5. 保鲜膜
    6. 注射器1毫升或2毫升
    7. 纸巾
    8. 培养皿
    9. 猎鹰管
    10. Eppendorf管
    11. 移液器吸头
    12. 无菌牙签
    13. 注射器过滤器0.22μm

  2. 生物材料
    1. 拟南芥NahG种子(Lawton等,1995)
    2. 携带具有感兴趣基因的双元载体的根癌土壤杆菌菌株(LBA4404,GV3101或C58C1)

  3. 试剂
    1. 无菌去离子水
    2. 甘油(CARLO ERBA试剂,目录号:453752)
    3. NaCl(AppliChem,目录号:121659.1210)
    4. 胰蛋白胨(Biolife,目录号:412290)
    5. 酵母提取物(AppliChem,目录号:403687.1210)
    6. 细菌琼脂(MICROKIT,目录号:BCB006 +)
    7. MES(2-(N-吗啉代)乙磺酸)(Sigma-Aldrich,目录号:M2933)
    8. MgCl 2(AppliChem,目录号:131396.1210)
    9. DMSO(Sigma-Aldrich,目录号:M81802)
    10. 乙酰丁香酮(Sigma-Aldrich,目录号:D134406)
    11. 利福平(Duchefa Biochemie,目录号:R0146.0005)
    12. 四环素(Sigma-Aldrich,目录号:T3383)
    13. 庆大霉素(Sigma-Aldrich,目录号:G3632)
    14. 壮观霉素(Duchefa Biochemie,目录号:S0188.0005)
    15. 卡那霉素(Sigma-Aldrich,目录号:K4378)
    16. LB培养基(见食谱)
    17. 1 M MES(见食谱)
    18. 1 M MgCl 2(见食谱)
    19. 0.1 M乙酰丁香酮(见食谱)
    20. 渗透解决方案(请参阅食谱)
    21. 抗生素溶液(取决于构建体和农杆菌菌株,参见食谱)

设备

  1. 无菌锥形瓶
  2. 在短日照条件下(8小时光照/ 16小时黑暗)能够维持21℃的植物生长室,具有140-150μmolm-2秒-1光强度(Radiber SA,产品目录号:AGP-1400)
  3. 能够进行OD 600测量的分光光度计,诸如Shimadzu UV-1601(Shimadzu,目录号:206-67001-34)
  4. 自动P1000,P200和P20微量移液器
  5. 培养箱设置在28°C,例如孵化器D-6450哈瑙(Heraeus Instruments,型号:D-6450)
  6. 能够保持28℃和180rpm的培养箱振荡器,如New Brunswick TM I26(Eppendorf,New Brunswick scientific,型号:I26,目录号:M1324-0000)
  7. 离心50毫升管,如Rotofix 32A(Hettich,目录号:1206-01)
  8. 高压灭菌器

程序

  1. 植物生长条件
    注:拟南芥NahG植物是获得令人满意的瞬时基因表达/蛋白表达的关键步骤。在该方案中,使用约4周龄的拟南芥NahG植物用于农杆菌渗入。
    1. 将种子放在浸有水的土壤中,在植物盆中混合。用保鲜膜覆盖锅并将其置于21°C下8小时光照/ 16小时黑暗周期的生长室中。
    2. 直到幼苗有两片真叶(约7-10天)。

    3. 小心地将苗移植到40个穴位的种子托盘中的最终目的地。

    4. 在生长室内种植植物约3周,在21°C下8小时光照/ 16小时黑暗周期。
    5. 当植物具有10-12叶的坚固花环并且没有开始开花时,植物准备好渗透。

  2. 农杆菌文化
    注意:该方案可以与至少三种不同的农杆菌常用菌株:LBA4404,GV3101和C58C1一起使用。
    1. 使用甘油原液和无菌牙签,将农杆菌克隆条纹用于补充有适当抗生素的LB固体培养板中。将培养板置于28°C培养箱中培养48小时,以获得新鲜和单菌落。
    2. 在开始渗透前一天,使用平板上的新菌落在LB液体培养基中开始液体农杆菌培养物。使用无菌牙签从单菌落中挑选土壤杆菌生物质,将其置于无菌锥形瓶中,所述无菌锥形瓶具有补充有适当抗生素的20ml LB液体培养基,并在28℃和180rpm下培养过夜。

  3. 渗透
    1. 农杆菌制备
      1. 将饱和的培养物倒入50ml Falcon管中。
        4,000 em x g 旋转细胞10分钟。
      2. 通过滗析弃去LB培养基上清液。消除尽可能多的上清液。

      3. 使用1体积的新鲜制备的渗透缓冲液,用涡旋重悬细胞沉淀。
      4. 重新悬浮后,在室温下将培养物在黑暗中放置2-4小时。
      5. 准备饱和培养物的1/20稀释液,测量OD 600并计算必需体积以具有0.05的最终OD 600。用渗透缓冲液稀释。
    2. 渗透程序
      1. 用重悬浮培养物填充1或2毫升无针注射器,最终OD600值为0.05。通过在叶的远轴侧按压注射器(无针)进行渗透,同时用指尖在近轴侧施加反压。如果渗透成功,观察液体在叶片内的扩散情况。
        渗透整个叶子( ca。 100μl细菌悬浮液/离开)。

      2. 使用薄纸干燥叶面上多余的培养物(图1,视频1)。
      3. 浸润后两到四天,观察浸润蛋白的荧光或收获浸润的叶子以进行蛋白质提取。


        图1.瞬时表达的实验步骤 NahG 拟南芥 使用根癌土壤杆菌(Agrobacterium tumefaciens)的植物A.携带所需构建体的含有农杆菌的Eppendorf管1ml注射器,4周龄的NahG拟南芥属植物和一张纸。 B.植物渗透(详见程序部分)。

        视频1

笔记


  1. 一些叶子在渗入细菌悬液后可能会发生坏死
  2. 在长日照条件下(16小时光照/ 8小时黑暗周期)生长NahG拟南芥,如果植物没有开始开花,可能不会影响瞬时表达。然而,当植物在短日照条件下生长时,植物叶子更大,更容易渗入。
  3. 也可以先调整OD 600,然后在黑暗中培养2-4小时。
  4. 每株植物渗入不超过4-5片叶子。当更多的叶子渗入时,植物的存活率显着减少。
  5. 使用这个协议,蛋白质积累的水平在渗透后3天是最佳的。
  6. 实际上玫瑰花结的任何一片叶子都可以渗透出积极的结果,但最年长的叶子往往表现出较低的信号,而最年轻的叶子往往对细菌接种表现出较强的反应,伴随着坏死。
  7. 干燥过量的培养物对于渗透的成功并不是决定因素,但由于叶子在潮湿时粘在一起会引起较高程度的坏死,因此非常值得推荐。

食谱

  1. LB培养基(1 L)
    NaCl 5克
    胰蛋白胨10克
    酵母提取物5克
    (仅用于固体培养基)细菌琼脂16克
  2. 1 M MES(100毫升)
    17.5克MES
    完成无菌去离子水
    调整pH值至5.6
    通过过滤消毒
    在室温下储存
  3. 1 M MgCl 2(100毫升)
    20.3克MgCl 2 2/2 完成无菌去离子水
    通过高压灭菌来消毒
    在室温下储存
  4. 0.1M乙酰丁香酮(10毫升)
    0.196克乙酰丁香酮
    10毫升DMSO
    准备1毫升等分试样,并将它们储存在-20°C。
  5. 渗透液(100毫升)

    加满无菌去离子水
    注意:在使用前准备渗透液。
  6. 抗生素解决方案

致谢

作者感谢Huang Tan在成像过程中的技术帮助。这项工作得到了西班牙科学技术大学(AGL2016-75819-C2)和中国科学院上海植物逆境生物学中心的支持。该协议源自以前的出版物(Rosas-Díaz等人,2017)。作者声明他们没有利益冲突。

参考

  1. Krenek,P.,Samajova,O.,Luptovciak,I.,Doskocilova,A.,Komis,G。和Samaj,J。(2015年)。 农杆菌介导的瞬时植物转化:原理,方法和应用。 Biotechnol Adv 33(6 Pt 2):1024-1042。
  2. Lawton,K.,Weymann,K.,Friedrich,L.,Vernooij,B.,Uknes,S.and Ryals,J。(1995)。 拟南芥中的系统获得性抗性需要水杨酸,而不是乙烯。 Mol Plant Microbe Interact 8(6):863-870。
  3. Rosas-Díaz,T.,Cana-Quijada,P.,Amorim-Silva,V.,Botella,M.A.,Lozano-Duran,R.和Bejarano,E.R。(2017)。 拟南芥NahG 植物作为一种合适且高效的瞬时表达系统, Agrobacterium tumefaciens 。 Mol Plant 10(2):353-356。
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引用:Cana-Quijada, P., Bejarano, E. R., Lozano-Durán, R. and Rosas-Díaz, T. (2018). Transient Expression Assay in NahG Arabidopsis Plants Using Agrobacterium tumefaciens. Bio-protocol 8(12): e2894. DOI: 10.21769/BioProtoc.2894.
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