Materials and Reagents

Pipette tips
Scalpel
Filter paper (Whatman ﬁlters) (No. 1)
0.22 μm Millex-GV ﬁlter (Merck, catalog number: SLGP033NS )
Amicon Ultra-15 Centrifugal Filter Unit 3KD (Merck, catalog number: UFC900308 )
P. sojae strain (P6497)
V8 juice (https://www.campbells.com/v8/vegetable-juice/)
Calcium carbonate (CaCO₃) (Sigma-Aldrich, catalog number: 398101 )
Potassium Dihydrogen Phosphate (KH₂PO₄) (Merck, catalog number: 1048730250 )
Yeast extract (Sigma-Aldrich, catalog number: Y1625-1KG )
Magnesium sulfate heptahydrate (MgSO₄•7H₂O) (Sigma-Aldrich, catalog number: RES0089M-A702X )
D-glucose (Sigma-Aldrich, catalog number: G8270-100G )
L-asparagine (Sigma-Aldrich, catalog number: A0884-100G )
β-sitosterol (Sigma-Aldrich, catalog number: S1270-10MG )
Thiamine(VB1) (Sigma-Aldrich, catalog number: T4625-5G )
Milli-Q-ﬁltered H₂O
Ammonium sulfate (NH₄)₂SO₄(Sigma-Aldrich, catalog number: A4418-100G )
Tris-aminomethane (Tris-HCl) (Sigma-Aldrich, catalog number: 154563-1KG )
Ethylene Diamine Tetraacetic Acid (EDTA) (Sigma-Aldrich, catalog number: 80849-10MG )
30% acrylamide
10% sodium dodecyl sulfate (SDS) (Sigma-Aldrich, catalog number: L3771-100G )
10% ammonium persulfate (Sigma-Aldrich, catalog number: A3678-25G )
N,N,N',N'-Tetramethylethylenediamine (TEMED) (Sigma-Aldrich, catalog number: T9281-25ML )
Coomassie brilliant blue (CBB-R-250) (Beyotime Biotechnology, catalog number: P0017 )
Ethanol (ALADDIN-E, catalog number: A112719 )
Acetic acid (ALADDIN-E, catalog number: A116174 )
Methanol (ALADDIN-E, catalog number: M116127 )
V8 juice with 2% agar (see Recipes)
Synthesis liquid medium (see Recipes)
TE buffer (see Recipes)
SDS-PAGE Gel (see Recipes)
R-250 staining solution (see Recipes)
Destaining solution (see Recipes)

Equipment

Pipettes
Conical flask (1 L)
Beaker (5 L)
Measuring cylinder (1 L)
Constant temperature incubator (CTI) (25 °C)
Constant temperature shaker (CTS) (25 °C, 100 rpm)
Centrifuge
Liquid chromatograph (GE Healthcare, model: ÄKTA avant 25 )
Column Sephadex G-25S (GE Healthcare, catalog number: 17140801 )
Chromatography column (GE Healthcare, model: Superdex^TM 200 Increase, catalog number: 28990944 )

Note: The materials, reagents and equipment not provided company and catalog number can be ordered from any qualified company for using in this experiment.

Procedure

Preparation and culture of P. sojae for secreted protein
1. P. sojae strain P6497 was grown in sterilized 10% V8 juice with 2% agar medium at 25 °C in the Constant Temperature incubator (CTI) under dark conditions for 4 days (Figure 1).
  
  Figure 1. P. sojae colony grown on V8 plates for 4 days
2. For secreted proteins production, cut P. sojae grown on V8 plates into 3 mm x 3 mm discs with a sterilized scalpel. Transfer twenty P. sojae discs into 1 L synthesis liquid medium and incubate on the Constant temperature shaker (CTS) (25 °C, 100 rpm) for 10 days (Figure 2).
  
  Figure 2. P. sojae cultured in synthesis liquid medium for 10 days
Extraction and collection of secreted protein from medium by P. sojae
1. Collect P. sojae culture fluid by centrifugation at 1,200 x g for 20 min and ﬁlter through two layers of Whatman ﬁlters (No. 1) and then a 0.22 μm Millex-GV filter.
2. Add 70 g of (NH₄)₂SO₄ per 100 ml filtrate for precipitation and incubate for 12 h at 4 °C.
3. Dissolve the precipitate in 4 ml TE buffer.
Chromatographic puriﬁcation and isolation of extracts secreted by P. sojae (see the Video 1)

Video 1. Gel filtration desalting chromatography for separating the precipitated proteins by AKTA
1. Gel filtration desalting chromatography for separating the precipitated proteins
  1. Equilibrate the Sephadex G-25S, the bound material and elute system using TE buffer.
  2. Load the protein samples into the Column Sephadex G-25S.
  3. Collect the fractions corresponding to each absorbance peak respectively and concentrate using 3-kD molecular mass centrifugal ﬁlter devices. (The absorption peaks stand for the line of UV280 on the screen of AKTA, Figure 3)
2. Gel filtration sieving chromatography
  1. Collect the eluted samples from Sephadex G-25S and subsequently inject into TE buffer-equilibrated Superdex^TM 200 Increase chromatography column.
  2. Collect the fractions corresponding to each absorbance peak respectively, and then desalt and concentrate using 3-kD molecular mass centrifugal ﬁlter devices (see Amicon Ultra-15 Centrifugal Filter Unit 3-kD manual).
3. SDS-PAGE analysis of the fractions corresponding to each absorbance peak
  1. Load the concentrated protein (20 μl about 100 μg/ml) into 10% SDS-PAGE gel and subject to electrophoresis for 1 h in 100 V.
  2. Stain the gel with Coomassie Brilliant Blue overnight and then destain for 3 h at room temperature.
4. Separation and elution of protein in SDS-PAGE Gel
  1. For the fractions showing more than one band after Coomassie Brilliant Blue staining in the SDS-PAGE gel (Figure 3), cut each protein band from gel and into 2-mm segments. Dry it (remove the water with Filter paper).
  2. After drying, dissolve these segments in 0.1 ml 0.1% SDS (pH 6) and incubate at 70 °C for 1 h and then 37 °C overnight.
  3. Collect the supernatant by centrifugation at 12,000 x g for 10 min at 4 °C.
    
    Figure 3. SDS-PAGE analysis of the protein fractions. A. Absorbance peaks corresponding to the fractions in B, respectively. B. Fractions of secreted protein show on SDS-PAGE gel after Coomassie Brilliant Blue staining.
Identification of secreted protein by mass spectrometry
Analyze the collected supernatant by LC-MS/MS as described before (Wang et al., 2003; Ma et al., 2015). Proteins matched with peptides identified by mass spectrometry are used for further research.
Note: For further analysis of these proteins, they were expressed in Pichia pastoris (Km71) using the pPIZ9k vector or Escherichia coli (Rosetta) using pET32a.

Data analysis

Based on the molecular mass, secreted proteins were separated by Gel filtration sieving chromatography. Protein with similar molecular mass will form one peak. The area of the peak represents the abundance of purified proteins. Proteins collected in each peak were subsequently used for LC-MS/MS analysis and the data were shown in Ma et al. (2015).

Notes

Kanamycin (50 μg/μl) can be added in the medium in case of bacterial pollution.
(NH₄)₂SO₄ should be added into the medium slowly with continuous swirling.

Recipes

V8 juice with 2% agar (1 L)
V8 juice: 100 ml
CaCO₃: 1 g
ddH₂O: 900 ml
Agar power: 20 g
Synthesis liquid medium (1 L)
0.5 g KH₂PO₄
0.5 g Yeast extract
0.25 g MgSO₄•7H₂O
0.001 g thiamine
25 g Glucose
1 g Asparagines
0.01 g β-Sitosterol
Add water to a volume of 900 ml
Mix and make up to 1 L with Milli-Q-ﬁltered H₂O
TE buffer
10 mM Tris-HCl (pH 7.5) with 1 mM EDTA (TE)
10% SDS-PAGE Gel
1. Resolving gel buffer (10 ml):
  4 ml Milli-Q-ﬁltered H₂O
  3.3 ml 30% acrylamide (Bio-Rad)
  2.5 ml 1.5 M Tris-HCl (Sigma-Aldrich) (pH 8.8)
  0.1 ml 10% SDS (Sigma-Aldrich)
  0.1 ml 10% ammonium persulfate (Sigma-Aldrich)
  0.004 ml TEMED (Bio-Rad)
2. Stacking gel buffer (3 ml):
  2.1 ml Milli-Q-ﬁltered H₂O
  0.5 ml 30% acrylamide (Bio-Rad)
  0.38 ml 1.5 M Tris-HCl (Sigma-Aldrich) (pH 6.8)
  0.03 ml 10% SDS (Sigma-Aldrich)
  0.03 ml 10% ammonium persulfate (Sigma-Aldrich)
  0.003 ml TEMED (Bio-Rad)
R-250 staining solution (100 ml)
Methanol: 45 ml
ddH₂O: 45 ml
Acetic acid: 10 ml
R-250 Coomassie: 0.25 g
Destaining solution (1 L)
Ethanol: acetic acid: ddH₂O = 1:1:2

Acknowledgments

The authors would like to thank Dr. Zhenchuan Ma and this work was fund by grants to Yuanchao Wang from the China National Funds for Innovative Research Groups (Grant No. 31721004), the Special Fund for Agro-scientific Research in the Public Interest (201303018), and the China Agriculture Research System (CARS-004-PS14).

Competing interests

The authors declare that there is no conflict of interest.

References

Ma, Z., Song, T., Zhu, L., Ye, W., Wang, Y., Shao, Y., Dong, S., Zhang, Z., Dou, D., Zheng, X., Tyler, B. M. and Wang, Y. (2015). A Phytophthora sojae glycoside hydrolase 12 protein is a major virulence factor during soybean infection and is recognized as a PAMP. Plant Cell 27(7): 2057-2072.
Wang, Y. C., Hu, D. W., Zhang, Z. G., Ma, Z. C., Zheng, X. B. and Li, D. B. (2003). Purification and immunocytolocalization of a novel Phytophthora boehmeriae protein inducing the hypersensitive response and systemic acquired resistance in tobacco and Chinese cabbage. Physiol Mol Plant Pathol 63: 223-232.