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Extraction of Chloroplast Proteins from Transiently Transformed Nicotiana benthamiana Leaves
从瞬时转化的本氏烟草中提取叶绿体蛋白质   

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Abstract

This rapid protocol allows the extraction of chloroplast enriched proteins from Nicotiana benthamiana (N. benthamiana) leaves that were transiently transformed to express an epitope tagged protein of interest. Thus, it can serve as a tool to study the chloroplastidic localization of the protein of interest when it is combined with western-blot analysis.
Agrobacterium-mediated transformation (Agroinfiltration, Romeis et al., 2001) is used to transiently express a protein carrying an epitope tag in tobacco leaves. Here, co-infiltration with an Agrobacterium strain harboring 19 K from soil-borne wheat mosaic virus suppresses posttranscriptional gene silencing and therefore increases transformation efficiency (Te et al., 2005).
The chloroplast isolation of the transformed leaves is based with modifications on Romeis et al. (2001), and includes mechanical breakage of cell wall and membranes, the removal of unbroken tissue by filtration and the separation of intact chloroplasts by centrifugation through a Percoll layer.

Materials and Reagents

  1. Nicotiana benthamiana plants
  2. Agrobacterium tumefaciens (A. tumefaciens) strain GV3101 carrying a recombinant binary plasmid with the gene of interest fused to an epitope tag
  3. Agrobacterium tumefaciens strain GV3101 harboring K19 (Te et al., 2005)
  4. Tryptone (AppliChem GmbH, catalog number: 403682 .1210)
  5. Yeast extract (AppliChem GmbH, catalog number: A1552 , 0500)
  6. Sucrose (AppliChem GmbH, catalog number: A2211 , 0500)
  7. MgSO4 (Merck KGaA, catalog number: 105886 )
  8. Appropriate antibiotic
  9. Agar (Carl Roth, catalog number: 2266.2 )
  10. Acetosyringone (Sigma-Aldrich, catalog number: D134406 )
  11. MES buffer (Carl Roth, catalog number: 4256.3 )
  12. Liquid nitrogen
  13. Percoll (GE healthcare, catalog number: 17-0891-01 )
  14. EDTA (Carl Roth, catalog number: 8040.1 )
  15. DTT (AppliChem GmbH, catalog number: A2948 )
  16. MgCl2 (Merck KGaA, catalog number: 105833 )
  17. Glycerol (AppliChem GmbH, catalog number A3739 ,0500)
  18. Hepes (Carl Roth, catalog number: 9105 )
  19. Sorbitol (Merck KGaA, catalog number: 56755 )
  20. Serum bovine albumin (Carl Roth, catalog number 8076.2 )
  21. Protease inhibitor cocktail Complete Mini EGTA-free (Roche Diagnostics, catalog number: 11 836 153 001 )
  22. YEP medium (see Recipes)
  23. Agromix buffer (see Recipes)
  24. Isolation buffer (see Recipes)
  25. Protein extraction buffer (see Recipes)

Equipment

  1. 1 ml syringe (Terumo Medical Corporation, catalog number: BS-01H )
  2. Centrifuge cooled at 4 °C
  3. 47 μm nylon mesh (Carl Roth, catalog number: XA63.1 )
  4. 2 ml glass homogenizer with round bottom shape (A. Hartenstein, catalog number: HOG2 )
  5. Light microscope with phase contrast condenser and phase contrast compatible objective lens, such as Objective LD A-Plan 40x/0.5 Ph2 (ZEISS)
  6. Incubator shaker
  7. Rotary shaker
  8. Photospectrometer for OD600
  9. 1.5 ml reaction tubes (SARSTEDT AG, catalog number 72.706 )
  10. 50 ml conical centrifuge tubes (SARSTEDT AG, catalog number 62.548.004 )

Procedure

  1. Transient transformation of N. benthamiana
    1. Agrobacterium tumefaciens GV3101 strain harboring 19 K and Agrobacterium tumefaciens GV3101 strain carrying the recombinant binary plasmid for the transient expression of the gene of interest (with epitope tag) were streaked from glycerol stocks on YEP Agar plates (containing the appropriate antibiotics), respectively. Plates were incubated at 28 °C for 48 h to obtain single colonies.
    2. Single colonies from plates not older than 1 week were inoculated into 15 ml YEP medium (containing the appropriate antibiotics) in 50 ml tubes and incubated at 28 °C overnight at 180 rpm in an shaker.
    3. The next day the overnight cultures (OD600 between 1.0 and 1.5) were centrifuged at 2,500 x g for 10 min at room temperature. The supernatant was discarded.
    4. Each pellet was re-suspended in 5 ml of agromix buffer.
    5. The Agromix was incubated for 2 h in the dark at 120 rpm on a rotary shaker at room temperature.
    6. Both bacterial suspensions (19 K, gene of interest) were combined such that the final suspension with an OD600 between 0.5 and 1.0 consisted of 20% agrobacteria with 19 K and of 80% agrobacteria carrying the recombinant binary plasmid for the transient expression of the gene of interest in total.
      Note: This ratio for 19K in the infiltration mixture has been proven to be effective to enhance the transient expression of several proteins due to the suppression of gene silencing by 19K. However, the optimum ratio might be different for other proteins. An OD600 higher than 1.0 in the infiltration mixture could lead to effects of toxicity in tobacco.
    7. This mix was infiltrated by pressing the tip of a syringe without needle onto the lower leaf surface of 3-4 weeks old, well watered, Nicotiana benthamiana. The third, fourth and fifth youngest leaf were used for agroinfiltration (Figure 1A). Every leaf half was once injected with 100-150 µl of the mix (see Video 1). The infiltrated area on the leaves were marked using a marker pen (Figure 1B).
      Note: It is important to water the plants in the morning of the day on which the agroinfiltration takes place. Otherwise, it is difficult to infiltrate the leaf tissue successfully without damaging too much the tissue.

    Video 1. Agroinfiltration procedure

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  2. Chloroplast isolation of transiently transformed N. benthamiana
    Preliminary remark: For optimal yield of intact chloroplasts the plant material should be harvested from tobacco plants at the end of the dark cycle and kept in the dark to avoid excess levels of starch accumulation that can rupture the chloroplast envelope during centrifugation. In addition, all protocol steps are carried out at 4 °C.
    1. 100 mg of plant material of transiently transformed N. benthamiana leaves, 2-3 days after agro-infiltration, were homogenized in 400 μl of ice-cold isolation buffer using a precooled glass homogenizer.
    2. The homogenate was filtered through a 47 μm nylon mesh and kept on ice and in the dark.
    3. Preparation of 40% Percoll layer: 132 µl of Percoll were mixed thoroughly with 198 µl of isolation buffer and the layer of 40% Percoll was deposited in the bottom of a 1.5 ml reaction tube.
    4. 400 µl of the filtered chloroplast containing homogenate was placed carefully on top of the 40% Percoll layer and centrifuged at 7,000 x g at 4 °C for 1 min. The intact chloroplasts will sediment as a green pellet, whereas the broken chloroplasts remain on the top of the Percoll layer (Figure 2).
      Notes:
      1. Alternatively, the centrifugation of intact chloroplasts can also be performed more gently at 1,700 x g for 6 min at 4 °C.
      2. The supernatant was discarded and the chloroplasts were re-suspended gently in a small volume (20-30 µl) of isolation buffer. The chloroplast enriched suspension was kept on ice.
    5. The quality of the pelleted chloroplasts was confirmed by applying phase contrast microscopy. Intact chloroplasts appear opaque with a halo surrounding them (Figure 3A). Broken chloroplasts exhibit a dark green, granular, and non-refractive appearance (Figure 3B).
    6. Subsequently, the chloroplasts were centrifuged at 1,000 x g at 4 °C for 5 min to pellet the chloroplasts and the supernatant was removed and discarded.
    7. Chloroplasts were shock frozen in liquid nitrogen.
    8. The freeze-fractured chloroplasts were re-suspended in 50µl of ice-cold protein extraction buffer and centrifuged at 15,000 x g at 4 °C for 10 min.
    9. The supernatant (with soluble chloroplastidic proteins) was transferred to a new tube.
    10. The pellet, enriched with non-soluble chloroplastidic proteins, was re-suspended in 50 µl of ice-cold protein extraction buffer.
    11. Samples could either be stored at -70 °C or directly be used for western blot analyis to compare the protein expression in the chloroplast fraction versus total crude extract from transiently transformed tobacco leaves.
      Note: The protein yield for isolation of chloroplastidic proteins from 100 mg leaf material is in the range of 10 and 20 µg in total. 5-10 µg can be used for the western blot analysis.

Representative data



Figure 1. Agroinfiltration procedure.
A. The leaves indicated with number 3, 4 and 5 are best used for agroinfiltration. B. The infiltrated areas are marked with a marker pen.


Figure 2. Separation of intact from broken chloroplasts after centrifugation through a 40% Percoll layerl



Figure 3. A Intact chloroplasts and B broken chloroplast visualized by phase contrast microscopy

Recipes

  1. YEP medium
    0.5 % tryptone (m/v)
    0.5% yeast extract (m/v)
    0.5% sucrose (m/v)
    50 mM MgSO4
    For plates: 1.5% agar (m/v) was added
    The YEP medium was autoclaved
  2. Agromix buffer (has to be prepared fresh)
    10 mM MES (adjusted to pH=5.8 with 1 M KOH)
    10 mM MgCl2
    150 μM acetosyringone
    In purified (deionized and autoclaved) water
  3. Isolation buffer (has to be prepared fresh)
    0.33 M Sorbitol
    50 mM Hepes (adjusted with 1 M KOH to pH=7.0)
    0.1% (m/v) serum bovine albumin
    2 mM EDTA
    1 mM MgCl2
    In purified (deionized and autoclaved) water
  4. Protein extraction buffer (has to be prepared fresh)
    10% (m/v) glycerol (from autoclaved stock solution)
    5 mM EDTA (from autoclaved stock solution)
    10 mM DTT
    100 mM HEPES (adjusted with 1 M KOH to pH=7.2, sterilfiltrated)
    Protease inhibitor cocktail
    In purified (deionized and autoclaved) water

Acknowledgments

This protocol was adapted from previous work (Romeis et al., 2001; Te et al., 2005; Pineda et al., 2010). I would like to acknowledge R. Deeken (Department of Molecular Plant Physiology and Biophysics, University of Wuerzburg, Germany) and the DFG Research Training Group 1342 (University of Wuerzburg, Germany) for funding. In addition, I would like to than C.W. Lee for help and advice.

References

  1. Pineda, M., Sajnani, C. and Baron, M. (2010). Changes induced by the Pepper mild mottle tobamovirus on the chloroplast proteome of Nicotiana benthamiana. Photosynth Res 103(1): 31-45.
  2. Romeis, T., Ludwig, A. A., Martin, R. and Jones, J. D. (2001). Calcium-dependent protein kinases play an essential role in a plant defence response. EMBO J 20(20): 5556-5567.
  3. Te, J., Melcher, U., Howard, A. and Verchot-Lubicz, J. (2005). Soilborne wheat mosaic virus (SBWMV) 19K protein belongs to a class of cysteine rich proteins that suppress RNA silencing. Virol J 2: 18. 

简介

该快速方案允许从瞬时转化以表达感兴趣的表位标记蛋白的本氏烟草(本氏烟草)叶中提取富含叶绿体的蛋白。因此,当它与Western印迹分析结合时,它可以作为研究目标蛋白质的叶绿体定位的工具。使用土壤杆菌介导的转化(Agroinfiltration,Romeis等人,2001)瞬时表达在烟草叶中携带表位标签的蛋白质。在这里,与土壤传播的小麦花叶病毒携带19K的农杆菌菌株的共浸润抑制转录后基因沉默,并因此增加转化效率(Te等人,2005) 。转化的叶子的叶绿体分离基于Romeis等人(2001)的修改,包括细胞壁和膜的机械破坏,通过过滤去除未破坏的组织,通过Percoll层离心分离完整叶绿体。

材料和试剂

  1. 本尼特烟草植物
  2. 根瘤土壤杆菌(根瘤土壤杆菌)菌株GV3101,携带具有与表位标签融合的感兴趣基因的重组二元质粒
  3. 含有K19的土壤杆菌根瘤土壤杆菌菌株GV3101(Te等人,2005)
  4. 胰蛋白胨(AppliChem GmbH,目录号:403682.1210)
  5. 酵母提取物(AppliChem GmbH,目录号:A1552,0500)
  6. 蔗糖(AppliChem GmbH,目录号:A2211,0500)
  7. MgSO 4(Merck KGaA,目录号:105886)
  8. 合适的抗生素
  9. 琼脂(Carl Roth,目录号:2266.2)
  10. Acetosyringone(Sigma-Aldrich,目录号:D134406)
  11. MES缓冲液(Carl Roth,目录号:4256.3)
  12. 液氮
  13. Percoll(GE healthcare,目录号:17-0891-01)
  14. EDTA(Carl Roth,目录号:8040.1)
  15. DTT(AppliChem GmbH,目录号:A2948)
  16. MgCl 2(Merck KGaA,目录号:105833)
  17. 甘油(AppliChem GmbH,目录号A3739,0500)
  18. Hepes(Carl Roth,目录号:9105)
    液氮
  19. Percoll(GE healthcare,目录号:17-0891-01)
  20. EDTA(Carl Roth,目录号:8040.1)
  21. DTT(AppliChem GmbH,目录号:A2948)
  22. MgCl 2(Merck KGaA,目录号:105833)
  23. 甘油(AppliChem GmbH,目录号A3739,0500)
  24. Hepes(Carl Roth,目录号:9105)
    ... 液氮
  25. Percoll(GE healthcare,目录号:17-0891-01)
  26. EDTA(Carl Roth,目录号:8040.1)
  27. DTT(AppliChem GmbH,目录号:A2948)
  28. MgCl 2(Merck KGaA,目录号:105833)
  29. 甘油(AppliChem GmbH,目录号A3739,0500)
  30. Hepes(Carl Roth,目录号:9105)
    ......
  31. Light microscope with phase contrast condenser and phase contrast compatible objective lens, such as Objective LD A-Plan 40x/0.5 Ph2 (ZEISS)
  32. Incubator shaker
  33. Rotary shaker
  34. Photospectrometer for OD600
  35. 1.5 ml reaction tubes (SARSTEDT AG, catalog number 72.706)
  36. 50 ml conical centrifuge tubes (SARSTEDT AG, catalog number 62.548.004)

程序

  1. N的瞬态变换。本bent。
    1. 将携带19K和具有用于瞬时表达感兴趣的基因(具有表位标签)的重组双元质粒的根瘤土壤杆菌GV3101菌株的根瘤土壤杆菌 GV3101菌株从甘油原种在YEP琼脂平板(含有合适的抗生素)上。将板在28℃孵育48小时以获得单个菌落
    2. 将来自不大于1周的平板的单个菌落接种到50ml管中的15ml YEP培养基(含有合适的抗生素)中,并在振荡器中以180rpm在28℃下温育过夜。
    3. 第二天,将过夜培养物(1.0和1.5之间的OD 600)在室温下以2500xg离心10分钟。弃去上清液。
    4. 将每个沉淀重新悬浮在5ml的农杆菌缓冲液中
    5. 将Agromix在室温下在旋转振荡器上在黑暗中以120rpm温育2小时
    6. 将两种细菌悬浮液(19K,目的基因)组合,使得OD 600在0.5和1.0之间的最终悬浮液由20%的具有19K的土壤杆菌和80%的携带重组二元的农杆菌质粒用于目的基因的瞬时表达 注意:在浸润混合物中的19K的比率已经被证明是有效的,以增强几种蛋白质的瞬时表达,这是由于19K的基因沉默的抑制。然而,对于其他蛋白质,最佳比例可能不同。在渗透混合物中高于1.0的OD 600 可能会导致烟草中的毒性。
    7. 通过将没有针头的注射器的尖端压在3-4周龄,充分浇灌的烟草烟草的下叶面上来渗透该混合物。第三,第四和第五最年轻的叶用于农杆菌浸润(图1A)。每个叶子一半注入100-150微升的混合物(见视频1)。用记号笔标记叶子上的浸润区域(图1B) 注意:在进行农杆菌渗透的那一天早上对植物浇水是很重要的。否则,难以在不损害太多组织的情况下成功地渗透叶组织。

    视频1.农业过滤程序
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  2. 瞬时转化的N的叶绿体分离。 本bentiana
    初步评论:为了使完整叶绿体的最佳产量,植物材料应该在黑暗周期结束时从烟草植物中收获,并保持在黑暗中以避免过量水平的淀粉积聚,其可在离心期间破坏叶绿体包膜。 此外,所有方案步骤在4℃下进行。
    1. 100mg的瞬时转化的植物材料。 本生烟草叶,在农杆菌浸润后2-3天,使用预冷的玻璃匀浆器在400μl冰冷的分离缓冲液中匀浆。
    2. 将匀浆通过47μm尼龙网过滤,并保存在冰上和在黑暗中
    3. 40%Percoll层的制备:将132μlPercoll与198μl分离缓冲液充分混合,并将40%Percoll层沉积在1.5ml反应管的底部。
    4. 将400μl含有过滤的叶绿体的匀浆小心地置于40%Percoll层的顶部,并在4℃下以7000xg离心1分钟。完整的叶绿体将沉淀为绿色沉淀,而破碎的叶绿体保留在Percoll层的顶部(图2)。
      注意:
      1. 或者,完整叶绿体的离心也可以在4℃下以1,700×g更温和地进行6分钟。
      2. 弃去上清液,将叶绿体轻轻地重悬在小体积(20-30μl)的分离缓冲液中。将富含叶绿体的悬浮液保持在冰上。
    5. 通过应用相差显微镜检查证实了沉淀的叶绿体的质量。 完整叶绿体看起来是不透明的,其周围有晕环(图3A)。 破碎的叶绿体表现出深绿色,颗粒状和非折射的外观(图3B)。
    6. 随后,将叶绿体在1,000×g下在4℃离心5分钟以沉淀叶绿体,并除去上清液并弃去。
    7. 叶绿素在液氮中冲击冷冻
    8. 将冷冻断裂的叶绿体重悬浮于50μl冰冷的蛋白提取缓冲液中,并在4℃下以15,000×g离心10分钟。
    9. 将上清液(具有可溶性叶绿体蛋白)转移到新管中
    10. 将富含不溶性叶绿体蛋白的沉淀重悬于50μl冰冷的蛋白提取缓冲液中。
    11. 样品可以储存在-70℃或直接用于蛋白质印迹分析,以比较叶绿体部分中的蛋白质表达与瞬时转化的烟叶的总粗提取物。
      注意:从100mg叶材料分离叶绿体蛋白的蛋白质产量总共在10和20μg的范围内。 5-10μg可用于蛋白质印迹分析。

代表数据



图1.农杆菌渗入程序。 A.用编号3,4和5表示的叶子最适用于农杆菌浸润。 B.浸润区域用记号笔标记。


图2.通过40%Percoll层1 离心后,完整叶片与破碎叶绿体的分离

图3.通过相差显微镜观察到的完整叶绿体和B破碎的叶绿体

食谱

  1. YEP介质
    0.5%胰蛋白胨(m/v)
    0.5%酵母提取物(m/v) 0.5%蔗糖(m/v) 50mM MgSO 4 对于板:加入1.5%琼脂(m/v) 将所述YEP培养基高压灭菌
  2. Agromix缓冲液(必须新鲜制备)
    10mM MES(用1M KOH调节至pH = 5.8) 10mM MgCl 2/
    150μM乙酰丁香酮 在纯化(去离子和高压灭菌)的水中
  3. 隔离缓冲区(必须新鲜制备)
    0.33摩尔山梨醇 50mM Hepes(用1M KOH调节至pH = 7.0)
    0.1%(m/v)血清牛白蛋白 2mM EDTA 1mM MgCl 2
    在纯化(去离子和高压灭菌)的水中
  4. 蛋白提取缓冲液(必须新鲜制备)
    10%(m/v)甘油(来自高压灭菌的原液)
    5mM EDTA(来自高压灭菌的原液)
    10 mM DTT
    100mM HEPES(用1M KOH调节至pH = 7.2,经过无菌过滤) 蛋白酶抑制剂混合物
    在纯化(去离子和高压灭菌)的水中

致谢

该协议改编自以前的工作(Romeis等人,2001; Te等人,2005; Pineda等人,2010) 。 我要感谢R. Deeken(德国维尔茨堡大学分子植物生理学和生物物理系)和DFG研究培训组1342(德国维尔茨堡大学)的资助。 此外,我想要比李先生的帮助和建议。

参考文献

  1. Pineda,M.,Sajnani,C.and Baron,M。(2010)。 由辣椒轻度斑驳烟草花叶病毒在烟草的叶绿体蛋白质组上诱导的变化 本bent。 Photosynth Res 103(1):31-45。
  2. Romeis,T.,Ludwig,A.A.,Martin,R.and Jones,J.D。(2001)。 钙依赖性蛋白激酶在植物防御反应中发挥重要作用。 EMBO J 20(20):5556-5567
  3. Te,J.,Melcher,U.,Howard,A.and Verchot-Lubicz,J。(2005)。 土壤中小麦花叶病毒(SBWMV)19K蛋白属于一类抑制RNA沉默的富含半胱氨酸的蛋白质 。 Virol J 2:18. 
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Klinkenberg, J. (2014). Extraction of Chloroplast Proteins from Transiently Transformed Nicotiana benthamiana Leaves. Bio-protocol 4(18): e1238. DOI: 10.21769/BioProtoc.1238.
  2. Klinkenberg, J., Faist, H., Saupe, S., Lambertz, S., Krischke, M., Stingl, N., Fekete, A., Mueller, M. J., Feussner, I., Hedrich, R. and Deeken, R. (2014). Two fatty acid desaturases, STEAROYL-ACYL CARRIER PROTEIN Delta9-DESATURASE6 and FATTY ACID DESATURASE3, are involved in drought and hypoxia stress signaling in Arabidopsis crown galls. Plant Physiol 164(2): 570-583.
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