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）

Malou  Fraiture; Frédéric  Brunner

doi:10.21769/BioProtoc.1232

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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）

Malou Fraiture

FB Frédéric Brunner email

发布: 2014年09月05日第4卷第17期 DOI: 10.21769/BioProtoc.1232 浏览次数: 9608

评审: Ru ZhangAnonymous reviewer(s)

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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).

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.

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)

Materials and Reagents

Arabidopsis thaliana Col-0 purchased from ‘Nottingham Arabidopsis Stock Centre’
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]
Malt extract (Carl Roth, catalog number: X976.2 )
Peptone from casein (tryptic digest) (Carl Roth, catalog number: 8986.2 )
Agar-agar (bacteriological) (Carl Roth, catalog number: 2266.3 )
Ultrapure H₂O (type 1) (filtered with MilliQ Reference purification system) (Merck KGaA)
MES [2-(N-morpholino) ethanesulfonic acid] (Carl Roth, catalog number: 4256.3 )
KCl (Carl Roth, catalog number: P017.1 ) (2 M solution with ultrapure H₂O)
Ethanol (Merck KGaA, catalog number: 100983 ) (diluted to 20% with ultrapure H₂O)
XK16/20 chromatography column (GE Healthcare, catalog number: 28-9889-37 )
Sepharose SP Fast Flow (GE Healthcare, catalog number: 17-0729-01 ) (stored at 4 °C)
Source 15S 4.6/100 PE column (GE Healthcare, catalog number: 17-5182-01 ) (stored at 4 °C)
GS90 soil (CL Ton Kokos) (Gebrüder Patzer, catalog number: 10-00800-40 )
Vermiculite for plant culture (BayWa AG or Gebrüder Patzer)
Rubber stoppers (Carl Roth, catalog number EC93.1 )
Flg22 peptide from Pseudomonas aeruginosa (Felix et al., 1999) (synthesized, 50 µM, stored at -20 °C)
Malt-peptone-agar (see Recipes)
Malt-peptone medium (see Recipes)
Buffer A (see Recipes)
Buffer B (see Recipes)

Equipment

Petri dishes (94 x 16 mm, with vents) (Greiner Bio-One GmbH, catalog number: 633180 )
Paraffin film (Parafilm M) (Sigma-Aldrich, catalog number: P7793 )
Needle holder (Carl Roth, catalog number: 6189.1 )
Lancets (Carl Roth, catalog number: 6181.1 )
1 L culture flasks (erlenmeyer-shaped, with straight neck and metal caps) (SCHOTT AG, DURAN^®, catalog number: 21 771 54 )
Laboratory funnel (minimum 15 cm diameter) (Carl Roth, catalog number: YA51.1 )
Nylon mesh (60 µm pore size) (Carl Roth, catalog number: XA95.1 )
1 L laboratory bottles with screw-cap (SCHOTT AG, DURAN^®, catalog number: 21 801 54 5 )
Small silica gel bag
50 ml Falcon conical centrifuge tubes (VWR International, catalog number: 21008-940 )
0.2 µm bottle-top filters for 500 ml (Carl Roth, catalog number: AC20.1 )
Paper scissors
6 ml thick-walled glass test tubes (Carl Roth, catalog number: HA75.1 )
1 ml syringe (B. Braun Melsungen AG, Omnifix-F Tuberculin, catalog number: 9161406V )
Syringe needle (27 Ga. x ¾ in., size 20) (B. Braun Melsungen AG, Sterican, catalog number: 4657705 )
Spatula (Carl Roth, catalog number: YK51.1 )
1.5 ml reaction tubes with caps (VWR International, catalog number: 700-5239 )
MilliQ Reference purification system (Merck KGaA, catalog number: Z00QSV001 )
Laminar flow clean bench
15 °C incubator
Front-loading lyophilizer of at least 10 L capacity with basins (e.g. L10, WKF-Gesellschaft für elektrophysikalischen Apparatebau, not produced anymore)
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)
Vacuum pump (Gardner Denver, Welch, catalog number: 2522C-02 )
4 °C cold lab chamber or cold room
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)
Plant growth chamber (controlled light, temperature and humidity)
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

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如何引用

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.