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Modified Single-Cell Transient Gene Expression Assay in Barley Epidermal Cells
大麦表皮细胞中一种改良的单细胞转基因技术-粒子枪法   

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Abstract

Transient gene expression via biolistic particle delivery is a widely used technique for gene functional analysis in plants. In this protocol we describe a modified single-cell transient expression assay through transformation with a particle inflow gun of the model PDS-1000/He system (Bio-Rad). This assay was originally optimized for analyzing cell death activity and disease resistance function of the barley MLA (mildew locus A) disease resistance proteins against the powdery mildew fungus, which can be further adopted for other purposes for other types of plant proteins and in some other plant species, including Arabidopsis thaliana.

Keywords: Transient gene expression(瞬时基因表达), Single epidermal cell(单个表皮细胞), Particle bombardment(粒子轰击), DNA coating(DNA涂层), Barley, wheat(大麦、小麦)

Materials and Reagents

  1. Barley (Hordeum vulgare L.) plants, 1 week old seedlings
  2. Powdery mildew strain(s), fresh conidiospores as inoculum
  3. Benzimidazol (Genview, catalog number: 51-17-2 )
  4. Agar (Japan, plant cell culture tested)
  5. CaCl2 (Sigma-Aldrich, catalog number: C7902 )
  6. X-gluc (Inalco, catalog number: 1758-0600 )
  7. Coomassie Brilliant Blue R-250 (Amresco, catalog number: 0472 )
  8. Ethanol (Beijing Chemical Works)
  9. Glycerol (Beijing Chemical Works)
  10. Methanol (Beijing Chemical Works)
  11. Lactic acid (Beijing Chemical Works)
  12. Plasmid
  13. Reporter DNA
  14. K3Fe [CN6] (Sinopharm Chemical Reagent, catalog number: 10016718 )
  15. Triton X-100 (AMRESCO, catalog number: 0694 )
  16. Spermidine (Sigma-Aldrich, catalog number: S-4139 ) (see Recipes)
  17. GUS staining solution (see Recipes)
  18. Destaining solution (see Recipes)
  19. Benzimidazol plates (see Recipes)
  20. Coomassie blue solution (see Recipes)

Equipment

  1. PDS-1000/He delivery system (Bio-Rad Laboratones)
  2. Macrocarrier (Bio-Rad Laboratones, catalog number: 1652335 )
  3. Rupture disc (900 psi) (Bio-Rad Laboratones, catalog number: 1652328 )
  4. Centrifuges (Eppendorf, catalog number: 5424 )
  5. Fluorescence microscope (Carl Zeiss, Axio Scope. A1 )
  6. pH meter (Mettler Toledo, FE20K)
  7. Gold microcarrier: 1.0 μm in diameter (Bio-Rad Laboratones,catalog number: 165-2263 )

Procedure

  1. Preparations
    1. Inoculate plants with powdery mildew spores to prepare inoculum and sow barley seeds to grow plants for bombardment one week in advance. The plants were grown in a growth chamber under a 16 h/8 h, 20 °C/18 °C day/night cycle with 70% relative humidity.
    2. Prepare Benzimidazol plates one day in advance.
  2. Bombardment
    1. Cut primary leaves and put them on Benzimidazol plates with adaxial side up (Figure 1), 3-5 leaves per petridish (90 mm) per shot, incubate at least 4 h before shooting.


      Figure 1. Picture showing preparation of Benzimidazol agar plate with barley leaves for bombardment. 5-6 barley primary leaves were detached from 1 week old barley seedlings and put side by side with adaxial side up on prepared Benzimidazol agar plate.

    2. Prepare gold particles (20 shots):
      1. Weigh 9 mg gold particles in a 1.5 ml tube.
      2. Add 1 ml 70% ethanol, vortex 5 min, sediment particles for 15 min on bench.
      3. Spin 2 sec. (about 2,000 rpm), discard supernatant.
      4. Repeat 3 times: add 1 ml sterile H2O, vortex 2 min, sediment 1 min, spin 2 sec. (about 2,000 rpm), discard supernatant.
      5. Add 1 ml of 50% glycerol (in water), vortex (gold particles can be stored at -20 °C for 2-3 weeks).
    3. Coat the gold particles (use 50 μl gold particle solution for one shot):
      1. Vortex gold particle for at least 5 min.
      2. Mix equal molar plasmid and reporter DNA (e.g. GUS or GFP reporter), do not use more than 2 μg DNA in total, add ddH2O when volume is less than 5 μl.
      3. Aliquot 50 μl gold particles into each empty tube, then add DNA solution.
      4. While vortexing, add: 50 μl 2.5 M CaCl2 drop-by-drop, then 20 μl 0.1 M spermidine, vortex for 3 min in total.
      5. Sediment particles for 1 min, spin 2 sec. (2,000 rpm), discard supernatant.
      6. Add 140 μl 70% ethanol, vortex, spin 2 sec. (2,000 rpm), discard supernatant.
      7. Add140 μl 100% ethanol, vortex, spin 2 sec. (2,000 rpm), discard supernatant.
      8. Add 15 μl 100% ethanol, vortex, store on ice until used.
    4. Bombard, for each shot repeat the following steps:
      1. Fix the macrocarriers in macrocarier holder, suspend particles by pipetting, and apply the particles onto the macrocarrier. Dry on the bench.
      2. Dip rupture disc (900 psi) in 100% (v/v) 2-propanol and subsequentlyplace it into rupture disk retaining cap, add few more drops of 2-propanol.
      3. Insert macrocarrier holder with stop-screen in stop screen holder at position 1 (from top) (Figure 2).


        Figure 2. Picture showing the PDS-1000/He delivery system. Indicated are position 1 and 3 that reserved for macrocarrier holder and patridish holder, respectively.

      4. Insert petridish with leaves at position 3 (Figure 2).
      5. Apply vacuum up to 27 inches of mercury, trigger the shot.
      6. Arrange leaves on the petridish, put in incubator.
        Note: We put the leaves on the dish side by side with adaxial side up (Figure 1).
    5. (Omit this step if fungal inoculation is not necessary)
      Inoculate with powdery mildew condiospores at least 4 h after bombardment.
  3. For GFP index scoring
    36-48 h after bombardment count GFP expressing cell numbers using fluorescence microscope.
    Note: The total number of cells here is the sum of compatible (haustorium, seccondary hyphae) and incompatible (only appressorium) on GUS expressing cells. We score all of the five leaves and at least 60 cells were scored.
  1. For Fungal Haustorium index scoring
    1. 48 h after fungal spores inoculation
      Stain leaves for GUS expression: put leaves into 15 ml falcon tube containing about 8 ml X-gluc staining solution, vacuum infiltrate 5 min for 3 times, and incubate overnight to 24 h at 37 °C.
    2. 1 day after GUS staining
      Remove GUS staining solution, add about 10 ml destaining solution, store at RT at least 2 days.
    3. When time available:
      Stain for the fungus:
      1. Transfer leaves to large volume of ddH2O for 1 h.
      2. Stain in coomassie solution for few seconds.
      3. Wash twice in water.
      4. Mount on microscope slide in 50% glycerol. Once on the slide the samples should be scored within few days.
    4. Score compatible (visible intracellular haustorium, and sometimes secondary hyphae on leaf surface) and incompatible (only fungal appressorium) interaction cell/site for GUS expressing cells.

Recipes

  1. Benzimidazol plates
    1% agar in water with 85 μM Benzimidazol (from 8.5 mM stock solution in water, 100 x)
    Note: The pH value for these plates is about 6.5. It is not necessary to adjust the pH value.
  2. Spermidine
    0.1 M solution, 1 g solution mix with 67.8 ddH2O, filter sterilized.
    Aliquot and stored at -20 °C (note: it's very hygroscopic and air sensitive, close and put back to freezer immediately when done.)
    2.5 M CaCl2 in water, sterile filtrate store in room temperature
  3. GUS staining solution
    0.1 M Na2HPO4/NaH2PO (pH 7.0)
    10 mM Na-EDTA
    5 mM K4Fe[CN6]
    5 mM K3Fe[CN6]
    0.1% Triton X-100 (v/v)
    20% methanol (v/v)
    1 g/L X-gluc
    adjust to pH 7.0
  4. Destaining solution: stock solution
    50% glycerol
    25% lactic acid
    25% H2O
    Dissolve 1 volume stock solution in 2 volumes ethanol.
  5. Coomassie blue solution
    0.6% coomassie blue (w/v) in 100% (v/v) methanol/or ethanol

Acknowledgments

This protocol is adapted from Shirasu et al. (1999); Shen et al. (2012) and Bai et al. (2012).

References

  1. Bai, S., Liu, J., Chang, C., Zhang, L., Maekawa, T., Wang, Q., Xiao, W., Liu, Y., Chai, J., Takken, F. L., Schulze-Lefert, P. and Shen, Q. H. (2012). Structure-function analysis of barley NLR immune receptor MLA10 reveals its cell compartment specific activity in cell death and disease resistance. PLoS Pathog 8(6): e1002752.
  2. Shen, Q. H., Zhou, F., Bieri, S., Haizel, T., Shirasu, K. and Schulze-Lefert, P. (2003). Recognition specificity and RAR1/SGT1 dependence in barley Mla disease resistance genes to the powdery mildew fungus. Plant Cell 15(3): 732-744.
  3. Shirasu, K., Nielsen, K., Piffanelli, P., Oliver, R., and Schulze-Lefert, P. (1999). Cell-autonomous complementation of mlo resistance using a biolistic transient expression system. Plant J 17: 293-299.

简介

通过生物射弹颗粒递送的瞬时基因表达是广泛使用的用于植物中基因功能分析的技术。 在该协议中,我们描述了通过用PDS-1000/He系统(Bio-Rad)的粒子流入枪转化的修饰的单细胞瞬时表达测定。 该测定最初被优化用于分析大麦MLA(霉菌基因座A)疾病抗性蛋白质对白粉菌真菌的细胞死亡活性和疾病抗性功能,其可以进一步用于其他目的用于其他类型的植物蛋白质和其他一些 植物物种,包括拟南芥(Arabidopsis thaliana)。

关键字:瞬时基因表达, 单个表皮细胞, 粒子轰击, DNA涂层, 大麦、小麦

材料和试剂

  1. 大麦(Hordeum vulgare L.)植物,1周龄幼苗
  2. 白粉病菌,新鲜分生孢子作为接种物
  3. 苯并咪唑(Genview,目录号:51-17-2)
  4. 琼脂(日本,植物细胞培养试验)
  5. CaCl 2(Sigma-Aldrich,目录号:C7902)
  6. X-gluc(Inalco,目录号:1758-0600)
  7. 考马斯亮蓝R-250(Amresco,目录号:0472)
  8. 乙醇(北京化工厂)
  9. 甘油(北京化工厂)
  10. 甲醇(北京化工厂)
  11. 乳酸(北京化工)
  12. 质粒
  13. 记者DNA
  14. (Sinopharm Chemical Reagent,目录号:10016718)
  15. Triton X-100(AMRESCO,目录号:0694)
  16. 亚精胺(Sigma-Aldrich,目录号:S-4139)(参见Recipes)
  17. GUS染色溶液(见配方)
  18. 解决方案(参见配方)
  19. 苯并咪唑板(见配方)
  20. 考马斯蓝溶液(见配方)

设备

  1. PDS-1000/He输送系统(Bio-Rad Laboratones)
  2. Macrocarrier(Bio-Rad Laboratones,目录号:1652335)
  3. 破裂片(900psi)(Bio-Rad Laboratones,目录号:1652328)
  4. 离心机(Eppendorf,目录号:5424)
  5. 荧光显微镜(Carl Zeiss,Axio Scope.A1)
  6. pH计(Mettler Toledo,FE20K)
  7. 金微载体:直径1.0μm(Bio-Rad Laboratones,目录号:165-2263)

程序

  1. 准备
    1. 用白粉菌孢子接种植物以制备接种物和母猪大麦种子,以提前一周生长用于轰击的植物。 将植物在生长室中在具有70%相对湿度的16小时/8小时,20℃/18℃日/夜循环下生长。
    2. 提前一天准备苯并咪唑板。
  2. 轰击
    1. 切下初级叶,将它们放在苯并咪唑板上,近轴侧向上(图1),每个培养皿每个培养皿3-5个叶片(90mm),孵育至少4小时,然后拍摄。

      图1.显示具有用于轰击的大麦叶的苯并咪唑琼脂板的制备图。将5-6个大麦初级叶与1周龄大麦分离 幼苗并肩并肩放在一起准备 苯并咪唑琼脂板。

    2. 准备金粒子(20张):
      1. 在1.5ml管中称量9mg金颗粒。
      2. 加入1ml 70%乙醇,涡旋5分钟,沉积颗粒在台上15分钟
      3. 旋转2秒。 (约2000rpm),弃去上清液
      4. 重复3次:加入1ml无菌H 2 O,涡旋2分钟,沉淀1分钟,旋转2秒。 (约2000rpm),弃去上清液
      5. 加入1ml 50%甘油(在水中),涡旋(金颗粒可以在-20℃下储存2-3周)。
    3. 涂覆金颗粒(使用50μl金颗粒溶液一次性):
      1. 漩涡金颗粒至少5分钟。
      2. 混合等摩尔质粒和报告基因(例如 GUS或GFP报告基因),不要使用超过2μg的DNA,当体积小于5时,加入ddH 2 O μl。
      3. 等分50μl金颗粒到每个空管,然后加入DNA溶液
      4. 在涡旋的同时,加入:50μl2.5M CaCl 2缓冲液,然后20μl0.1M亚精胺,总共涡旋3分钟。
      5. 沉淀颗粒1分钟,旋转2秒。 (2,000rpm),弃去上清液
      6. 加入140μl70%乙醇,涡旋,旋转2秒。 (2,000rpm),弃去上清液
      7. 加入140μl100%乙醇,涡旋,旋转2秒。 (2,000rpm),弃去上清液
      8. 加入15μl100%乙醇,涡旋,存储在冰上,直到使用。
    4. Bombard,对于每个镜头重复以下步骤:
      1. 将大载体固定在大载体支架中,通过吸移悬浮颗粒,并将颗粒应用于大载体上。 在长凳上干燥。
      2. 在100%(v/v)2-丙醇中浸渍破裂盘(900psi),随后将其置于破裂盘保持盖中,再加几滴2-丙醇。
      3. 在位置1(从顶部)(图2)将带有止动屏幕的宏载体支架插入止动屏支架中

        图2.显示PDS-1000/He输送系统的图片。显示的是分别为宏载波持有者和patridish持有者保留的位置1和3。
      4. 在叶片的位置3插入培养皿(图2)。
      5. 使用真空度高达27英寸汞柱,触发镜头
      6. 在培养皿上放置叶子,放在孵化器中。
        注意:我们把叶子放在碟子上并排放置,正面朝上(图1)。
    5. (如果不需要真菌接种,则省略此步骤)
      在轰击后至少4小时接种白粉病菌
  3. 用于GFP指数评分
    轰击后36-48小时使用荧光显微镜计数GFP表达细胞数 注意:这里的细胞总数是GUS表达细胞上的相容性(吸尘器,间隔菌丝)和不相容(仅吸附)的总和。 我们对所有五片叶进行评分,至少60个细胞得分。
  1. 用于真菌Haustorium指数评分
    1. 真菌孢子接种后48小时 用于GUS表达的染色叶:将叶子放入含有约8ml X-gluc染色溶液的15ml falcon管中,真空渗入5分钟3次,并在37℃下孵育过夜至24小时。
    2. GUS染色后1天
      取出GUS染色溶液,加入约10ml脱色液,室温下保存至少2天。
    3. 可用时间:
      真菌污渍:
      1. 转移到大体积的ddH 2 O 2保持1小时。
      2. 在考马斯溶液中染色几秒钟。
      3. 在水中洗两次。
      4. 在显微镜载玻片上在50%甘油中。 一旦在幻灯片上,样品应该在几天内得分。
    4. 分数相容(可见细胞内吸尘器,有时在叶表面上的次生菌丝)和不相容(仅真菌附着)相互作用细胞/GUS表达细胞的位点。

食谱

  1. 苯并咪唑板
    1%琼脂在水中,含有85μM苯并咪唑(来自8.5mM水中的储备溶液,100x) 注意:这些板的pH值为约6.5。 不需要调节pH值。
  2. 亚精胺
    0.1M溶液,1g溶液与67.8ddH 2 O混合,过滤灭菌。
    等分并储存在-20°C(注意:它是非常吸湿和空气敏感,关闭,并立即回到冰箱完成后)。
    2.5M CaCl 2水溶液,无菌滤液在室温下保存
  3. GUS染色溶液
    0.1M Na 2 HPO 4水溶液/NaH 2 PO 4水溶液(pH 7.0)
    10mM Na-EDTA
    5mM K 4 Fe [CN 6]
    5mM K 3 Fe [CN 6]
    0.1%Triton X-100(v/v) 20%甲醇(v/v) 1 g/L X-gluc
    调整至pH7.0
  4. 脱色溶液:储液
    50%甘油 25%乳酸
    25%H 2 O * 将1体积储备溶液溶解在2体积乙醇中。
  5. 考马斯蓝溶液
    0.6%考马斯蓝(w/v)在100%(v/v)甲醇/或乙醇中

致谢

该协议改编自Shirasu等人(1999); Shen等人(2012)和Bai等人(2012)。

参考文献

  1. Bai,S.,Liu,J.,Chang,C.,Zhang,L.,Maekawa,T.,Wang,Q.,Xiao,W.,Liu,Y.,Chai,J.,Takken,FL,Schulze -Lefert,P。和Shen,QH(2012)。 大麦NLR免疫受体MLA10的结构功能分析揭示了其在细胞死亡和疾病中的细胞区室比活性 PLoS Pathog 8(6):e1002752。
  2. Shen,QH,Zhou,F.,Bieri, S.,Haizel,T.,Shirasu,K.and Schulze-Lefert, (2003)。 大麦Mla病抗性基因对白粉病真菌的识别特异性和RAR1/SGT1依赖性。 a> 15(3):732-744。
  3. Shirasu,K.,Nielsen,K.,Piffanelli,P.,Oliver,R。,和Schulze-Lefert,P。(1999)。 单元格自主互补 mlo 使用生物射弹瞬时表达系统的抗性。 Plant J 17:293-299
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Copyright: © 2013 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. Bai, S., Chang, C., Han, X. and Shen, Q. (2013). Modified Single-Cell Transient Gene Expression Assay in Barley Epidermal Cells. Bio-protocol 3(9): e690. DOI: 10.21769/BioProtoc.690.
  2. Bai, S., Liu, J., Chang, C., Zhang, L., Maekawa, T., Wang, Q., Xiao, W., Liu, Y., Chai, J., Takken, F. L., Schulze-Lefert, P. and Shen, Q. H. (2012). Structure-function analysis of barley NLR immune receptor MLA10 reveals its cell compartment specific activity in cell death and disease resistance. PLoS Pathog 8(6): e1002752.
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