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Axenic Culture of Sclerotinia sclerotiorum and Preparation of Sclerotinia Culture Filtrate Elicitor 1 (SCFE1)-containing Fractions, Triggering Immune Responses in Arabidopsis thaliana
无菌培养核盘菌并从其培养液中分离引发拟南芥免疫反应的激发子(SCFE1)   

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Abstract

The necrotrophic white mold fungus Sclerotinia sclerotiorum (S. sclerotiorum) is pathogenic to a broad range of plant species, including the Brassicaceae model plant Arabidopsis thaliana (Boland and Hall, 1994; Bolton et al., 2006). In Arabidopsis thaliana (A. thaliana), the semi-purified proteinaceous S. sclerotinorum elicitor SCFE1 (Sclerotinia culture filtrate elicitor 1) is sensed at the plasma membrane by the receptor-like protein RLP30 and triggers strong immune responses (Zhang et al., 2013), similar to the bacterial elicitor flagellin (Felix et al., 1999). Elicitation of plant defenses with SCFE1 is a tool to dissect the signaling pathway involving RLP30 and to study immunity to necrotrophic fungi. Here, we describe a simple protocol to axenically grow S. sclerotiorum. Further, we present a two-step liquid chromatography-based method for the partial purification of SCFE1 from culture filtrate (Figure 1A-B). Measurement by gas chromatography of the emission of the plant stress hormone ethylene is proposed as a bioassay to monitor elicitor activity in the fractions throughout the purification procedure (Figure 1C).

Keywords: Sclerotinia sclerotiorum(油菜菌核病), Elicitor(诱导子), Cation exchange chromatography(阳离子交换色谱法), Ethylene assay(乙烯法), Axenic culture(无菌培养)


Figure 1.  Two-step chromatographic fractionation of S. sclerotiorum culture filtrate to obtain semi-purified SCFE1. A. Purification scheme of SCFE1. Crude filtrate (CF) is loaded onto a Sepharose SP cation exchange chromatography column. The eluate (S1) is diluted 10-fold and loaded onto a Source 15S cation exchange chromatography column. Elution is performed with a linear gradient of 0 to 0.3 M KCl and elution fractions of 0.5 ml (F1 - F100) are collected. FT = flow-through. B. Chromatogram of the SCFE1-containing fractions eluted from a Source 15S cation exchange chromatography column. The black line represents the protein elution profile monitored with OD280 nm. The grey line shows the increasing conductivity of the elution buffer. ma.u. = milli-arbitrary units. mS = milli-Siemens. C. Ethylene response in Arabidopsis Col-0 to SCFE1-containing fractions obtained by two-step cation exchange chromatography. Arabidopsis Col-0 leaf pieces are treated with 15 µl CF, undiluted and 10-fold diluted S1 as well as the Source 15S elution fractions (Fx). Treatment with 0.5 µM flg22 is used as a positive control for ethylene production. No treatment and treatment with 15 µl buffers A and B are used as negative controls. In this representative purification, SCFE1 is contained in fractions F24 to F52. Bars represent average values (n=2) ± S.D.

Materials and Reagents

  1. Arabidopsis thaliana Col-0 purchased from ‘Nottingham Arabidopsis Stock Centre’
  2. Sclerotinia sclerotiorum strain 1946 [purchased from the biological resource center ‘Deutsche Sammlung von Mikroorganismen und Zellkulturen’ (DSMZ) GmbH, shipped in a sterile culture tube on agar medium, stored at 15 °C in the dark]
  3. Malt extract (Carl Roth, catalog number: X976.2 )
  4. Peptone from casein (tryptic digest) (Carl Roth, catalog number: 8986.2 )
  5. Agar-agar (bacteriological) (Carl Roth, catalog number: 2266.3 )
  6. Ultrapure H2O (type 1) (filtered with MilliQ Reference purification system) (Merck KGaA)
  7. MES [2-(N-morpholino) ethanesulfonic acid] (Carl Roth, catalog number: 4256.3 )
  8. KCl (Carl Roth, catalog number: P017.1 ) (2 M solution with ultrapure H2O)
  9. Ethanol (Merck KGaA, catalog number: 100983 ) (diluted to 20% with ultrapure H2O)
  10. XK16/20 chromatography column (GE Healthcare, catalog number: 28-9889-37 )
  11. Sepharose SP Fast Flow (GE Healthcare, catalog number: 17-0729-01 ) (stored at 4 °C)
  12. Source 15S 4.6/100 PE column (GE Healthcare, catalog number: 17-5182-01 ) (stored at 4 °C)
  13. GS90 soil (CL Ton Kokos) (Gebrüder Patzer, catalog number: 10-00800-40 )
  14. Vermiculite for plant culture (BayWa AG or Gebrüder Patzer)
  15. Rubber stoppers (Carl Roth, catalog number EC93.1 )
  16. Flg22 peptide from Pseudomonas aeruginosa (Felix et al., 1999) (synthesized, 50 µM, stored at -20 °C)
  17. Malt-peptone-agar (see Recipes)
  18. Malt-peptone medium (see Recipes)
  19. Buffer A (see Recipes)
  20. Buffer B (see Recipes)

Equipment

  1. Petri dishes (94 x 16 mm, with vents) (Greiner Bio-One GmbH, catalog number: 633180 )
  2. Paraffin film (Parafilm M) (Sigma-Aldrich, catalog number: P7793 )
  3. Needle holder (Carl Roth, catalog number: 6189.1 )
  4. Lancets (Carl Roth, catalog number: 6181.1 )
  5. 1 L culture flasks (erlenmeyer-shaped, with straight neck and metal caps) (SCHOTT AG, DURAN®, catalog number: 21 771 54 )
  6. Laboratory funnel (minimum 15 cm diameter) (Carl Roth, catalog number: YA51.1 )
  7. Nylon mesh (60 µm pore size) (Carl Roth, catalog number: XA95.1 )
  8. 1 L laboratory bottles with screw-cap (SCHOTT AG, DURAN®, catalog number: 21 801 54 5 )
  9. Small silica gel bag
  10. 50 ml Falcon conical centrifuge tubes (VWR International, catalog number: 21008-940 )
  11. 0.2 µm bottle-top filters for 500 ml (Carl Roth, catalog number: AC20.1 )
  12. Paper scissors
  13. 6 ml thick-walled glass test tubes (Carl Roth, catalog number: HA75.1 )
  14. 1 ml syringe (B. Braun Melsungen AG, Omnifix-F Tuberculin, catalog number: 9161406V )
  15. Syringe needle (27 Ga. x ¾ in., size 20) (B. Braun Melsungen AG, Sterican, catalog number: 4657705 )
  16. Spatula (Carl Roth, catalog number: YK51.1 )
  17. 1.5 ml reaction tubes with caps (VWR International, catalog number: 700-5239 )
  18. MilliQ Reference purification system (Merck KGaA, catalog number: Z00QSV001 )
  19. Laminar flow clean bench
  20. 15 °C incubator
  21. Front-loading lyophilizer of at least 10 L capacity with basins (e.g. L10, WKF-Gesellschaft für elektrophysikalischen Apparatebau, not produced anymore)
  22. Floor-standing high-speed centrifuge (e.g. Sorvall RC-5B Plus Superspeed centrifuge and Sorvall SLA-1500 Superlite rotor with adapters for 50 ml Falcon conical centrifuge tubes, Thermo Fisher Scientific Inc., not produced anymore)
  23. Vacuum pump (Gardner Denver, Welch, catalog number: 2522C-02 )
  24. 4 °C cold lab chamber or cold room
  25. ÄKTA Explorer 10 liquid chromatography system (GE Healthcare, catalog number: 18-1300-00 ) equipped with:
    1. Pump P-900 
    2. Sample pump P-960 (GE Healthcare, catalog number: 18-6727-00 )
    3. Monitor UV-900 (GE Healthcare, catalog number: 18-1108-35 )
    4. Monitor pH/C-900 (GE Healthcare, catalog number: 18-1129-74 )
    5. Fraction collector Frac-950 (GE Healthcare, catalog number: 18-6083-00
    6. Rack A for 18/30 mm tubes (GE Healthcare, catalog number: 18-6083-11
    7. Rack B for 12 mm tubes (GE Healthcare, catalog number: 18-6083-12 )
    8. Unicorn control software (version 4.12 or more)
  26.  Plant growth chamber (controlled light, temperature and humidity)
  27.  Gas chromatograph with Al2O3-column and flame-ionization detector (e.g. GC-14A with analysis unit C-R4AX Chromatopac, Shimadzu Deutschland GmbH, not produced anymore)

Procedure

  1. Axenic culture of Sclerotinia sclerotiorum
    1. Work under sterile conditions when handling S. sclerotiorum cultures.
    2. Prepare fungal stock plate:
      1. Cut 2 agar plugs (0.5 x 0.5 cm) from the initial S. sclerotiorum-containing culture tube using a lancet.
      2. Place agar plugs in a Petri dish on malt-peptone-agar.
      3. Seal the plate with paraffin film.
      4. Let the fungus grow for 3 days at room temperature (RT) in the dark (Figure 2).
        Note: The mycelium should cover almost the whole surface of the plate.
      5. Store plate at 15 °C in the dark and reuse it for up to 3 months.
    3. Prepare fungal liquid culture:
      1. Prepare 1 L culture flasks containing 400 ml malt-peptone medium (25 flasks for 10 L culture).
      2. Cut agar plugs (0.5 x 0.5 cm) of fresh mycelium from the stock plate and inoculate each flask with 2 of them.
      3. Let fungus grow without shaking for 2-3 weeks at RT in the dark until the liquid surface is completely covered with mycelium.


    Figure 2. S. sclerotiorum growing on a malt-peptone-agar plate

  2. Freeze-drying of S. sclerotiorum culture filtrate
    1. Put a nylon mesh into a laboratory funnel. Filter the culture medium through the mesh and collect it.
    2. Discard the mycelium.
    3. Poor the culture filtrate into basins and freeze-dry it in a front-loading lyophilizer for 3 to 4 days.
    4. Store freeze-dried material at RT in a hermetically closed 1 L laboratory bottle. Add a silica gel bag to keep the material dry.

  3.  Re-suspension of the culture filtrate
    1. Re-suspend freeze-dried culture filtrate (e.g. 15 g dry weight) in a minimal volume (approx. 6 ml/g) of ice-cold buffer A in a 50 ml Falcon conical centrifuge tube.
    2. Centrifuge sample for 20 min at 10,000 x g and 4 °C.
    3. Collect the supernatant and keep on ice.
    4. Repeat steps C9-10 if necessary to remove all insoluble particles.
    5. Keep an aliquot of culture filtrate for subsequent activity assays in plants.
    6. Re-suspended culture filtrate may be stored at -20 °C.
      Note: Frozen concentrated culture filtrate is stable for at least one year. Repeated freeze-thaw cycles do not damage the sample.

  4.  Isolation of SCFE1: Rough fractionation by cation exchange chromatography
    1. For the isolation of SCFE1 from re-suspended culture filtrate, work with the ÄKTA Explorer liquid chromatography system cooled at 4 °C (in a cold lab chamber or cold room).
    2. All solutions for the chromatography should be prepared with ultrapure H2O, filtered through a bottle-top filter into clean, dust-free bottles and cooled at 4 °C.
    3. Pack a XK16/20 chromatography column from GE Healthcare with Sepharose SP Fast Flow matrix from GE Healthcare for cation exchange chromatography to around 15 ml column volume (CV).
      Note: For a small-scale purification (less than 5 g dry weight), 1 or 5 ml pre-packed Sepharose SP Fast Flow columns from GE Healthcare may be used.
    4. Wash column with 5 CV ultrapure H2O at a flow rate of 3 ml/min.
    5. Equilibrate column with 5 CV buffer A at a flow rate of 3 ml/min.
    6. Load re-suspended culture filtrate via the P-960 sample pump at a flow rate of 3 ml/min.
    7. Register flow, pressure, pH, conductivity, OD280 nm and OD215 nm during the run.
    8. Collect flow-through once OD280 nm increases and until loading is completed.
    9. Wash column with buffer A until OD280 nm and OD215 nm are back to the base line and stable.
    10. Elute in one step with 100% buffer B.
    11. Collect eluate into a 50 ml Falcon conical centrifuge tube using rack A for the fraction collector. Start collecting once OD280 nm exceeds 300 ma.u. (milli-arbitrary units) and stop when the end of the elution peak is reached.
    12. Wash column subsequently with 5 CV 2 M KCl, 5 CV ultrapure H2O, then 2 CV 20% ethanol for storage.
    13. Before proceeding to the second purification step, test activity of culture filtrate (CF), flow-through (FT) and eluate (S1) with the ethylene assay in Arabidopsis (see step F).
      Note: S1 should have the highest activity and contain most of SCFE1.
    14. Store S1 at 4 °C until next day or at -20 °C for more than 1 year. The flow-through may be discarded if the activity is successfully recovered in S1.

  5.  Isolation of SCFE1: Refined fractionation by cation exchange chromatography
    1. Dilute S1 10-fold with buffer A.
    2. Install a pre-packed Source 15S 4.6/100 PE column from GE Healthcare for cation exchange chromatography.
    3. Wash column with 5 CV ultrapure H2O at a flow rate of 1ml/min.
    4. Equilibrate column with 5 CV buffer A at a flow rate of 1 ml/min.
    5. Load total volume of diluted sample via the P-960 sample pump at a flow rate of 1 ml/min.
      Note: The pressure on the column increases with time. The flow rate may be reduced progressively.
    6. Register flow, pressure, pH, conductivity, OD280 nm and OD215 nm during the run.
    7. Collect flow-through once OD280 nm increases and until loading is completed.
    8. Wash column with buffer A until OD280 nm and OD215 nm are back to the base line and stable.
    9. Elute with a linear gradient from 0% to 60% buffer B in 40 CV at a flow rate of 0.5 to 1 ml/min (see Figure 1B for a typical elution profile).
    10. Collect 0.5 ml fractions in 1.5 ml reaction tubes using rack B for the fraction collector (approx. 100 fractions, labeled F1-F100). Create a program for automated fractionation with the method wizard of the Unicorn software.
      Note: SCFE1 should elute at a salt conductivity value between 8 and 16 mS/cm.
    11. Wash column with 5 CV 100% buffer B at a flow rate of 1 ml/min.
      Note: A small elution peak may appear. It does not need to be collected, as it does not contain any activity.
    12. Wash column with 5 CV water, then 2 CV 20% ethanol for storage.
      Note: If the pressure on the column remains higher than initially, it means that it was slightly clogged during sample loading and requires additional washing before reuse. In this case, follow the cleaning-in-place (CIP) procedure for Source 15S columns as described by the manufacturer.
    13. Test activity of flow-through (FT) and elution fractions (F1-F100) with the ethylene assay in Arabidopsis (see step F).
      Note: It is sufficient to test 1 fraction out of 5 for those with low OD280 nm and 1 fraction out of 2 for those corresponding to the elution peak.
    14. Store active fractions containing semi-purified SCFE1 at 4 °C for maximum 1 day or at -20 °C for more than 1 year. The flow-through may be discarded if the activity is successfully recovered in the elution fractions.

  6.  Ethylene assay
    1. Grow Arabidopsis Col-0 plants on soil (GS90/vermiculite mixture 3.5: 1) for 5 to 6 weeks in a growth chamber: 8 h light/16 h dark, 23 °C, 40–60% relative humidity, 100 µmol m-2 s-1 light intensity (short day conditions).
    2. Choose the biggest leaves from the plants. Cut each leaf into 6 rectangular pieces using paper scissors. Omit the edges, tip and stalk. Float leaf pieces on dH2O in a Petri dish (Figure 3).
      Note: Prepare the leaf pieces 12 to 20 h before elicitation. Leaf pieces left for a longer time will not respond well to elicitation anymore.
    3. Leave leaf pieces over night in the closed Petri dish at RT or in an air-conditioned room.
    4. Pipet 0.5 ml dH2O into glass test tubes. Use 2 or 3 tubes per sample to test (for duplicate or triplicate measurements).
    5. Put 3 leaf pieces into each test tube using a spatula.
      Note: Leaf pieces may rest for a few hours in the test tubes or may be elicited immediately.
    6. Add 5 to 15 µl of the samples to test (CF, FT, S1 and/or selected fractions of F1-F100). Include an untreated control and a positive control (5 µl of 50 µM flg22 = 0.5 µM final concentration).
    7. Close test tubes with rubber stopper and carefully shake tube rack to mix.
      Note: Leaves should stay in the water or on the surface but not stick to the glass.
    8. Incubate for 3 to 4 h at RT.
    9. Measure the ethylene content in each tube:
      1. Prick through the rubber stopper of a tube using a 1 ml syringe with a needle.
      2. Mix the air inside the tube by moving up and down several times.
      3. Withdraw 1 ml air and inject it into the gas chromatograph.
      4. Record retention time and peak area to determine the amount of ethylene in pmol/ml air (see Figure 1C for representative data sets).
        Note: For the untreated control, an average value of 0.60 +/- 0.21 pmol/ml air is expected. The mean values for buffer A and B are 0.81 +/- 0.23 and 0.81 +/- 0.23, respectively. With 1.30 +/- 0.20 pmol/ml, the ethylene production in the flg22 control is expected to be around 2x higher than in the non-treated and 1.5x higher than in the buffer controls. The values for culture filtrate (CF) can range from 2.5 to 15 pmol/ml. As culture filtrate yields a higher ethylene response than flg22, an active culture filtrate sample from a previous purification (stored at -20 °C) may be used as additional positive control.


    Figure 3. Cutting and floating of A. thaliana leaf pieces

Notes

  1. The ethylene assay is a very reliable and fast method to detect and quantify the immune-eliciting activity of the SCFE1-containing fractions. It also allows testing a large number of samples in a row. Other immune assays may however be used, such as the luminol-based oxidative burst assay to measure the production of reactive oxygen species (requires dialysis of the SCFE1-containing fractions to remove buffer), immunoblotting to detect activated MAP kinases or qRT-PCR for transcriptional profiling of defense-related genes (Zhang et al., 2013).
  2. To confirm that in the selected chromatographic fractions truly contain SCFE1 and no other or additional elicitor, the fractions may be tested on the Arabidopsis RLP30 knock-out mutants rlp30-1, -2 or -4. These mutants are completely insensitive to treatment with SCFE1-containing fractions, but react normally to flg22 challenge. Alternatively proteinase K sensitivity and heat stability of the immune-eliciting activity are good indicators for the identity of SCFE1 (Zhang et al., 2013).

Recipes

  1. Malt-peptone-agar (autoclaved)
    10 g malt extract
    2.5 g peptone from casein, tryptic digest
    15 g agar-agar
    dH2O qs. to 1 L
  2. Malt-peptone medium (autoclaved)
    10 g malt extract
    2.5 g peptone from casein, tryptic digest
    dH2O qs. to 1 L
  3. Buffer A
    0.1 M MES
    Ultrapure H2O
    Adjust pH to 5.4
    Sored at 4 °C
  4. Buffer B
    0.1 M MES
    0.5 M KCl
    Ultrapure H2O
    Adjust pH to 5.4
    Stored at 4 °C

Acknowledgments

We acknowledge Prof. Georg Felix and his group at the ZMBP for having established gas chromatography and the ethylene assay protocol in the research department.

References

  1. Boland, G. and Hall, R. (1994). Index of plant hosts of Sclerotinia sclerotiorum. Canadian J Plant Pathol 16(2): 93-108.
  2. Bolton, M. D., Thomma, B. P. and Nelson, B. D. (2006). Sclerotinia sclerotiorum (Lib.) de Bary: biology and molecular traits of a cosmopolitan pathogen. Mol Plant Pathol 7(1): 1-16.
  3. Felix, G., Duran, J. D., Volko, S. and Boller, T. (1999). Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. Plant J 18(3): 265-276.
  4. Zhang, W., Fraiture, M., Kolb, D., Löffelhardt, B., Desaki, Y., Boutrot, F. F., Tor, M., Zipfel, C., Gust, A. A. and Brunner, F. (2013). Arabidopsis receptor-like protein30 and receptor-like kinase suppressor of BIR1-1/EVERSHED mediate innate immunity to necrotrophic fungi. Plant Cell 25(10): 4227-4241.

简介

坏死性白霉菌真菌(Sclerotinia sclerotiorum)(核盘菌)对广泛的植物物种(包括十字花科模式植物拟南芥(Arabidopsis thaliana))具有致病性, (Boland和Hall,1994; Bolton等人,2006)。在拟南芥中(thaliana),是半纯化的蛋白质。通过受体样蛋白RLP30在质膜感测核盘菌诱导物SCFE1(核盘菌属培养物滤液诱发物1),并且引发强烈的免疫应答(Zhang等人。,2013),类似于细菌激发剂鞭毛蛋白(Felix等人,1999)。植物防御与SCFE1的诱导是一个工具,以解剖涉及RLP30的信号通路和研究对坏死性真菌的免疫。在这里,我们描述一个简单的协议来生长。菌核。此外,我们提出了一种两步液相色谱法从培养滤液中部分纯化SCFE1的方法(图1A-B)。通过气相色谱法测定植物应激激素乙烯的发射作为生物测定,以在整个纯化程序中监测级分中的激发剂活性(图1C)。

关键字:油菜菌核病, 诱导子, 阳离子交换色谱法, 乙烯法, 无菌培养


图1.  S的两步色谱分离。菌核培养滤液,得到半纯化的SCFE1。 A。 SCFE1的净化方案。将粗滤液(CF) Sepharose SP阳离子交换色谱柱。洗脱液(S1) 稀释10倍并加载到Source 15S阳离子交换器上 色谱柱。用线性梯度0进行洗脱 至0.3M KCl,并收集0.5ml(F1-F100)的洗脱级分。 FT =流通。 B.含SCFE1的级分的色谱图 从Source 15S阳离子交换色谱柱洗脱。的 黑色线代表用OD 280nm监测的蛋白质洗脱曲线。灰色线显示洗脱的增加的电导率 缓冲。 。 =毫 - 任意单位。 mS =毫西门子。 C.乙烯 拟南芥 Col-0对含有SCFE1的级分的反应 两步阳离子交换色谱。 拟南芥 Col-0叶片 用15μlCF,未稀释的和10倍稀释的S1以及 源15S洗脱级分(Fx)。用0.5μMflg22处理 用作乙烯生产的阳性对照。没有治疗和 用15μl缓冲液A和B处理用作阴性对照。在 这种代表性纯化,SCFE1包含在级分F24至 F52。 条表示平均值(n = 2)±S.D。

材料和试剂

  1. 拟南芥 Col-0从"诺丁汉拟南芥库存中心"购买,
  2. 核盘菌核菌菌株1946 [购自生物资源中心'Deutsche Sammlung von Mikroorganismen und Zellkulturen'(DSMZ)GmbH,在无菌培养管中在琼脂培养基上运输,在15℃下在黑暗中储存]
  3. 麦芽提取物(Carl Roth,目录号:X976.2)
  4. 来自酪蛋白的蛋白胨(胰蛋白酶消化物)(Carl Roth,目录号:8986.2)
  5. 琼脂(细菌学)(Carl Roth,目录号:2266.3)
  6. 超纯H 2 O(类型1)(用MilliQ参考纯化系统过滤)(Merck KGaA)
  7. MES [2-(N-吗啉代)乙磺酸](Carl Roth,目录号:4256.3)
  8. KCl(Carl Roth,目录号:P017.1)(具有超纯H 2 O的2M溶液)
  9. 乙醇(Merck KGaA,目录号:100983)(用超纯H 2 O稀释至20%)
  10. XK16/20色谱柱(GE Healthcare,目录号:28-9889-37)
  11. Sepharose SP Fast Flow(GE Healthcare,目录号:17-0729-01)(储存在4℃)
  12. Source 15S 4.6/100PE柱(GE Healthcare,目录号:17-5182-01)(保存在4℃)
  13. GS90土壤(CL Ton Kokos)(GebrüderPatzer,目录号:10-00800-40)
  14. 用于植物培养的蛭石(BayWa AG或GebrüderPatzer)
  15. 橡胶塞(Carl Roth,目录号EC93.1)
  16. (合成,50μM,储存于-20℃)的来源于铜绿假单胞菌(Pseudomonas)/绿脓杆菌的Flg22肽(Felix等人,1999) >
  17. 麦芽 - 蛋白胨 - 琼脂(见配方)
  18. 麦芽 - 蛋白胨培养基(见配方)
  19. 缓冲区A(参见配方)
  20. 缓冲液B(参见配方)

设备

  1. 培养皿(94×16mm,具有通风口)(Greiner Bio-One GmbH,目录号:633180)
  2. 石蜡膜(Parafilm M)(Sigma-Aldrich,目录号:P7793)
  3. 针架(Carl Roth,目录号:6189.1)
  4. 柳叶刀(Carl Roth,目录号:6181.1)
  5. 1L培养瓶(锥形,具有直颈和金属帽)(SCHOTT AG,DURAN ,目录号:21771 54)
  6. 实验室漏斗(最小直径15cm)(Carl Roth,目录号:YA51.1)
  7. 尼龙网(60μm孔径)(Carl Roth,目录号:XA95.1)
  8. 1L带有螺帽的实验室瓶(SCHOTT AG,DURAN ,目录号:21801554)
  9. 小硅胶袋
  10. 50ml Falcon锥形离心管(VWR International,目录号:21008-940)
  11. 用于500ml的0.2μm瓶顶过滤器(Carl Roth,目录号:AC20.1)
  12. 纸剪刀
  13. 6ml厚壁玻璃试管(Carl Roth,目录号:HA75.1)
  14. 1ml注射器(B.Braun Melsungen AG,Omnifix-F Tuberculin,目录号:9161406V)
  15. 注射器针(27 Ga,x 3/4英寸,尺寸20)(B.Braun Melsungen AG,Sterican,目录号:4657705)
  16. 铲子(Carl Roth,目录号:YK51.1)
  17. 1.5ml带盖的反应管(VWR International,目录号:700-5239)
  18. MilliQ参考纯化系统(Merck KGaA,目录号:Z00QSV001)
  19. 层流净化台
  20. 15℃培养箱
  21. 具有盆(例如,L10,WKF-gesellschaftfürelektrophysikalischen Apparatebau,不再生产)的至少10L容量的前负荷冻干机。
  22. 落地式高速离心机(例如 Sorvall RC-5B Plus Superspeed离心机和Sorvall SLA-1500 Superlite转头,带有用于50ml Falcon锥形离心管的适配器,Thermo Fisher Scientific Inc.,不再生产)
  23. 真空泵(Gardner Denver,Welch,目录号:2522C-02)
  24. 4°C冷室实验室或冷室
  25. ÄKTAExplorer 10液相色谱系统(GE Healthcare,目录号:18-1300-00),装备有:
    1. 泵P-900
    2. 样品泵P-960(GE Healthcare,目录号:18-6727-00)
    3. Monitor UV-900(GE Healthcare,目录号:18-1108-35)
    4. 监测pH/C-900(GE Healthcare,目录号:18-1129-74)
    5. 馏分收集器Frac-950(GE Healthcare,目录号:18-6083-00)
    6. 用于18/30mm管的架子A(GE Healthcare,目录号:18-6083-11)
    7. 用于12mm管的机架B(GE Healthcare,目录号:18-6083-12)
    8. Unicorn控制软件(4.12或更高版本)
  26.  植物生长室(受控光,温度和湿度)
  27. 具有Al 2 O 3 3柱和火焰离子化检测器(例如,具有分析单元C-R4AX Chromatopac的GC-14A)的气相色谱仪, Shimadzu Deutschland GmbH,不再生产)

程序

  1. 核盘菌的核酸培养物
    1. 在处理无菌条件下工作。 菌核文化。
    2. 准备真菌储存板:
      1. 从最初的S切割2个琼脂塞(0.5×0.5cm)。 含有培养物的培养管。
      2. 将琼脂塞置于培养皿中的麦芽 - 蛋白胨 - 琼脂上。
      3. 用石蜡膜密封板。
      4. 让真菌在黑暗中在室温(RT)下生长3天(图2) 注意:菌丝体应该覆盖板的几乎整个表面
      5. 将培养皿在15°C避光保存,并重复使用最多3个月。
    3. 准备真菌液体培养:
      1. 准备1升含有400ml麦芽 - 蛋白胨培养基的培养瓶(25个培养瓶用于10L培养)。
      2. 从原料板上切下琼脂块(0.5×0.5cm)的新鲜菌丝体,并用2个接种每个烧瓶。
      3. 使真菌在室温下在黑暗中不摇动生长2-3周,直到液体表面完全被菌丝体覆盖


    图2. S。 菌核生长 在麦芽 - 蛋白胨 - 琼脂平板上生长

  2. 冷冻干燥。 硬化培养物滤液
    1. 将尼龙网放入实验室漏斗中。 通过网过滤培养基并收集
    2. 丢弃菌丝体。
    3. 将培养物滤液倒入盆中,并在前置冷冻干燥器中冷冻干燥3至4天
    4. 将冷冻干燥的材料在室温下储存在密闭的1L实验室瓶中。 加入硅胶袋以保持材料干燥。

  3.  重新悬浮培养物滤液
    1. 在50ml Falcon锥形离心管中,在最小体积(约6ml/g)的冰冷缓冲液A中重新悬浮冷冻干燥的培养物滤液(例如15g干重) br />
    2. 在10,000×g和4℃下离心样品20分钟。
    3. 收集上清液并保持冰。
    4. 如果需要,重复步骤C9-10以除去所有不溶性颗粒
    5. 保留培养物滤液的等分试样用于随后在植物中的活性测定
    6. 重悬培养滤液可储存于-20℃。
      注意:冷冻浓缩培养滤液至少稳定一年。 重复的冻融循环不会损坏样品。

  4. SCFE1的分离:通过阳离子交换色谱的粗分级分离
    1. 为了从再悬浮的培养物滤液中分离SCFE1,使用冷却至4℃的ÄKTAExplorer液相色谱系统(在冷的实验室或冷室)进行操作。
    2. 用于色谱的所有溶液应当用超纯H 2 O制备,通过瓶顶过滤器过滤到干净,无尘的瓶中并在4℃下冷却。
    3. 包装来自GE Healthcare的XK16/20色谱柱,来自GE Healthcare的Sepharose SP Fast Flow基质,用于阳离子交换色谱至约15ml柱体积(CV)。
      注意:对于小规模纯化(小于5g干重),可以使用来自GE Healthcare的1或5ml预填充的Sepharose SP Fast Flow柱。
    4. 使用5CV超纯H 2 O以3ml/min的流速洗涤柱。
    5. 用5CV缓冲液A以3ml/min的流速平衡平衡柱
    6. 通过P-960样品泵以3ml/min的流速加载重新悬浮的培养物滤液
    7. 在运行期间记录流量,压力,pH,电导率,OD <280nm和OD <215> nm。
    8. 一旦OD <280> nm,收集流通量,直到装载完成
    9. 用缓冲液A洗涤柱,直到OD <280nm和OD <215nm,回到基线并稳定。
    10. 用100%缓冲液B一步洗脱
    11. 收集洗脱液到一个50毫升Falcon锥形离心管使用支架A的馏分收集器。开始收集一次OD <280> nm超过300 ma.u。 (毫当量单位),当达到洗脱峰的终点时停止
    12. 随后用5CV 2 M KCl,5CV超纯H 2 O,然后2 CV 20%乙醇洗涤柱,用于储存。
    13. 在进行第二纯化步骤之前,用拟南芥中的乙烯测定法(参见步骤F)测定培养物滤液(CF),流通液(FT)和洗脱液(S1)的测试活性。 > 注意:S1应该具有最高的活动性,并且包含大多数SCFE1。
    14. 将S1储存于4°C直至第二天或-20°C储存1年以上。如果活动在S1中成功恢复,流通可能会被丢弃。

  5.   SCFE1的分离:通过阳离子交换色谱精制分馏
    1. 用缓冲液A稀释S1 10倍
    2. 安装来自GE Healthcare的预包装Source 15S 4.6/100 PE柱进行阳离子交换层析
    3. 用5 CV超纯H 2 O以1ml/min的流速洗涤柱。
    4. 用5 CV缓冲液A以1ml/min的流速平衡柱
    5. 通过P-960样品泵以1 ml/min的流速加载稀释样品的总体积 注意:色谱柱上的压力随时间增加。流速可以逐渐减小。
    6. 在运行期间记录流量,压力,pH,电导率,OD <280nm和OD <215> nm。
    7. 一旦OD <280> nm,收集流通量,直到装载完成
    8. 用缓冲液A洗涤柱,直到OD <280nm和OD <215nm,回到基线并稳定。
    9. 在40℃以0℃至60%缓冲液B的线性梯度洗脱,流速为0.5至1ml/min(对于典型的洗脱曲线,参见图1B)。
    10. 使用rack B在级分收集器(约100个级分,标记为F1-F100)中在1.5ml反应管中收集0.5ml级分。使用Unicorn软件的方法向导创建用于自动分馏的程序。
      注意:SCFE1应该在8到16 mS/cm的盐电导率值下洗脱。
    11. 用5 CV 100%缓冲液B以1ml/min的流速洗涤柱。
      注意:可能出现小的洗脱峰。它不需要收集,因为它不包含任何活动。
    12. 用5 CV水洗涤柱,然后用2 CV 20%乙醇洗涤以储存 注意:如果色谱柱上的压力保持高于初始值,则表示在装载样品期间其轻微堵塞,需要在重新使用前进行额外清洗。在这种情况下,请按照制造商的说明,遵循Source 15S色谱柱的CIP清洗程序。
    13. 测试拟南芥中乙烯测定的流通(FT)和洗脱级分(F1-F100)的活性(参见步骤F)。
      注意:对于具有低OD 280nm的那些,测试5个中的1个部分就足够了,对于对应于洗脱峰的那些,测试2个中的1个部分就足够了。
    14. 将含有半纯化SCFE1的活性级分在4°C存储最多1天或在-20°C存储超过1年。如果活性在洗脱级分中成功回收,则可以丢弃流出物
  6.  乙烯测定
    1. 在生长室中将土壤上的拟南芥Col-0植物(GS90 /蛭石混合物3.5:1)生长5至6周:8小时光照/16小时黑暗,23℃,40-60%相对湿度,100μmolm-2 --s sup -1 -1光强度(短日条件)。
    2. 从植物中选择最大的叶子。用纸剪刀将每片切成6个矩形片。省略边缘,尖端和茎。在培养皿中的dH 2 O上漂浮叶片(图3) 注意:在引出前12到20小时准备叶片。留下较长时间的叶片将不再对诱导作出良好反应。
    3. 离开叶片过夜在封闭的培养皿在室温或在空调房间。
    4. 吸取0.5ml dH 2 O到玻璃试管中。每个样品使用2或3个试管进行测试(重复或重复测量)。
    5. 使用刮刀将3片叶片放入每个试管中。
      注意:叶片可能会在试管中静置几个小时,或者立即被引出。
    6. 加入5至15μl待测样品(CF,FT,S1和/或F1-F100的选定部分)。 包括未处理的对照和阳性对照(5μl的50μMflg22 =0.5μM终浓度)。
    7. 用橡胶塞关闭试管,小心摇动管架混合。
      注意:叶子应该留在水中或表面,但不要粘在玻璃上。
    8. 在室温下孵育3至4小时。
    9. 测量每个试管中的乙烯含量:
      1. 用1ml注射器用针刺穿管的橡胶塞。
      2. 通过上下移动几次来混合管内的空气。
      3. 取出1ml空气并将其注入气相色谱仪。
      4. 记录保留时间和峰面积以确定乙烯在pmol/ml空气中的量(对于代表性数据集,参见图1C)。
        注意:对于未处理的对照,预期平均值为0.60 +/- 0.21pmol/ml空气。缓冲液A和B的平均值分别为0.81 +/- 0.23和0.81 +/- 0.23。使用1.30 +/- 0.20pmol/ml,flg22对照中的乙烯产量预期比未处理的高2倍,并且比缓冲液对照高1.5倍。培养物滤液(CF)的值可以在2.5至15pmol/ml的范围内。因为培养物滤液产生比flg22更高的乙烯反应,所以来自之前纯化(储存在-20℃)的活性培养物滤液样品可用作另外的阳性对照。


    图3.切割和漂浮 A。 thaliana leaf pieces

笔记

  1. 乙烯测定是检测和定量含SCFE1级分的免疫引发活性的非常可靠和快速的方法。它还允许连续测试大量样品。然而可以使用其他免疫测定,例如基于鲁米诺的氧化爆发测定来测量活性氧的产生(需要透析含有SCFE1的级分以去除缓冲液),免疫印迹以检测活化的MAP激酶或qRT-PCR防御相关基因的转录谱(Zhang等人,2013)。
  2. 为了证实在所选的色谱级分中真正含有SCFE1而没有其它或另外的激发剂,可以在拟南芥RLP30敲除突变体rIp30-1 上测试所述级分, em> -2 或 -4 。这些突变体对含有SCFE1的级分的处理完全不敏感,但通常对flg22攻击反应。或者,蛋白酶K的敏感性和免疫引发活性的热稳定性是SCFE1的身份的良好指示(Zhang等人,2013)。

食谱

  1. 麦芽 - 蛋白胨 - 琼脂(高压灭菌)
    10克麦芽提取物
    2.5克来自酪蛋白,胰蛋白酶消化的蛋白胨
    15克琼脂 - 琼脂 dH 2 O qs。 到1 L
  2. 麦芽蛋白胨培养基(高压灭菌)
    10克麦芽提取物
    2.5克来自酪蛋白,胰蛋白酶消化的蛋白胨
    dH 2 O qs。 到1 L
  3. 缓冲区A
    0.1 M MES
    超纯H 2 O 2 / 将pH调节到5.4
    在4℃下烘干
  4. 缓冲区B
    0.1 M MES
    0.5 M KCl
    超纯H 2 O 2 / 将pH调节至5.4
    储存在4°C

致谢

我们感谢Georg Felix教授和他的团队在ZMBP已经在研究部门建立了气相色谱和乙烯测定方案。

参考文献

  1. Boland,G。和Hall,R。(1994)。 核盘菌的植物宿主索引。 a> Canadian J Plant Pathol 16(2):93-108
  2. Bolton,M.D.,Thomma,B.P.and Nelson,B.D。(2006)。 (Sclerotinia sclerotiorum)(Lib。)de Bary:生物学和分子特征一种世界性病原体。 Mol Plant Pathol 7(1):1-16。
  3. Felix,G.,Duran,J.D.,Volko,S。和Boller,T。(1999)。 植物对细菌的最保守结构域有敏感的感知系统鞭毛蛋白。植物J 18(3):265-276。
  4. 这些研究结果表明,这些研究结果表明,这些研究结果表明, 。 拟南芥受体样蛋白30和受体样激酶抑制剂BIR1- 1/EVERSHED调节对坏死性真菌的先天免疫。 植物细胞 25(10):4227-
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Copyright: © 2014 The Authors; exclusive licensee Bio-protocol LLC.
引用:Fraiture, M. and Brunner, F. (2014). Axenic Culture of Sclerotinia sclerotiorum and Preparation of Sclerotinia Culture Filtrate Elicitor 1 (SCFE1)-containing Fractions, Triggering Immune Responses in Arabidopsis thaliana. Bio-protocol 4(17): e1232. DOI: 10.21769/BioProtoc.1232.
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