Cell Fractionation and the Identification of Host Proteins Involved in Plant–Virus Interactions
Abstract
:1. Introduction
2. Cellular Organelles Are Involved in Virus Infection
3. Host Proteins Participate in Virus Infection
4. Changes in Accumulation and Subcellular Localization of Host Proteins
5. Potyviruses as a Model for Host–Viral Protein Interactions
6. Cellular Fractionation to Identify Host Proteins
Host | Virus | Host Protein | Viral Protein | Technique | Ref. |
---|---|---|---|---|---|
Transgenic tobacco BY-2 cells | ToMV | Sar1, Sec61, and TOM1 | 130 KDa and 180 KDa replication proteins | Membrane flotation analysis and Sucrose gradient sedimentation analysis | [20] |
Transgenic tobacco BY-2 cells | ToMV | Tm-1 | 130 K and 180 K | Differential centrifugation | [73] |
Cucumis sativus | ToRSV | N/A | NTB | Membrane fractionation | [74] |
Pea or lettuce plants | LMV | 20 s Proteasome | HCPro | 30% sucrose cushion and gel filtration column | [75] |
N. benthamiana | CiLV-C | N/A | P29, P15, MP, and P24 | Bimolecular Fluorescence Complementation combined with ultracentrifugation | [76] |
Tomato | TYLCV | HSP70 | CP | Sucrose density gradient | [77] |
N. benthamiana | PVA | Ck2, CPIP, HSP70, and CHIP | NIb, VPg, and CP | Membrane fractionation | [78] |
N. benthamiana | CMoV | SUMO1, SUMO2, and SCE1 | ORF4 | Cell wall fractionation | [79] |
7. The Process of Cellular Fractionation
Organelle | Marker Proteins | Tissue Type | Homogeniza-tion Buffer | Centrifugation Speed, Condition | Fraction Obtained | Gradient Centrifugation | Purification | Final Obtained Fraction | Ref. |
---|---|---|---|---|---|---|---|---|---|
Membrane fraction | See other organelles | Cucumis sativus leaves | Homogenization buffer one | 30,000× g, 30 min | Crude membrane (P30) (pellet) | 20–45% sucrose gradient centrifugate at 143,000× g for 4 h | - | Membranes separated into 13 fractions | [74] |
Cytoplasm | UDP-glucose pyrophosphorylase (UGPase) [83] | Tomato leaves | Nuclear extraction buffer | Filtrate centrifuged at 1300× g, 10 min | Cytoplasmic fraction concentrated 10 times by ultracentrifugation | 10–50% sucrose gradient centrifugation at 104,000× g for 20 h | - | 10 fractions obtained from the gradient | [77] |
Rice cell culture | Enzyme buffer | 100,000× g, 1 h | Remove top lipid layer, take supernatant | - | Add trichloroacetic acid to supernatant; centrifuge at 20,000× g for 5 min | Wash the pellet with cold acetone at 20,000× g for 15 min and take the pellet | [84] | ||
Vacuole | TIPs ( and isoforms), Epsilon subunit of tonoplast H+ ATPase (V-ATPase) [85] and [86] | Arabidopsis Rosette leaves | Protoplast solution | 80× g at 20 °C, 15 min | Pellet protoplast | 10% Ficoll buffer overlayed on 4% Ficoll and vacuole buffer; centrifugation at 50,000× g, 5 min at 10 °C | - | Vacuoles found on the 4% Ficoll buffer/vacuole buffer interface | [87] |
Chloroplast | Plastocyanin (PC) light harvesting complex b (LHC) [83,85] | N. benthamiana leaves | Enzyme mixture | 300× g, 16 min | Resuspend the pellet | 40% and 80% Percoll gradient and centrifugate at 3000× g, 25 min; collect chloroplasts at the interface of 40%/80% Percoll | - | Resuspend chloroplast in resuspension buffer | [83] |
ER | HDEL domain [85] | Castor Bean Endosperm | Homogenization buffer two | 1000× g, 15 min | supernatant | 20%, 30%, 40%, and 60% sucrose; centrifuge at 250,000× g, 22 h at 2 °C | Resuspend ER fraction between 20% and 30% and pellet via centrifugation at 250,000× g for 45 min | Resuspend ER pellet | [88] |
Mitochondria | Voltage-dependent, anion-selective channel protein 1-5 (VDAC1-5) [89] | Citrus pulp | Extraction buffer for mitochondria | 3000× g, 10 min then centrifuge the supernatant at 12,000× g, 15 min | Resuspend the pellet in washing buffer | 18%, 22.5%, and 35% Percoll gradient; ultracentrifugation at 50,000× g, 1 h | Mitochondrial band enriched at 22.5–35% Percoll gradient interface, then diluted with washing buffer, and centrifuged at 1500× g | Resuspend, purified mitochondria pellet in small volume of washing buffer | [90] |
Golgi | ADP-ribosylation factor 1 (ARF1) [91] | Wheat seedling | Extraction buffer for Golgi membranes | 3000× g, 20 min | supernatant | 25–40% sucrose gradient centrifuge at 100,000× g for 16 h | Ultracentrifuge fractions (1:10) at 100,000× g for 1 h | Resuspend the membrane pellet in 50 μL dilution buffer | [92] |
Nucleus | Histone H3 [93] | Tomato leaves | Nuclear extraction buffer | 1300× g, 10 min | Pellet | 10–50% sucrose gradient centrifuge at 104,000× g, 20 h | - | 10 Nuclei fractions | [77] |
Proteasomes | Regulatory Particle Triple-A ATPase subunit 2 (RPT2) and Regulatory Particle Non-ATPase 10 (RPN10) [94] | Arabidopsis seedlings | Extraction buffer for proteasomes | 30,000× g, 15 min | Supernatant | Precipitation with 2% and 10% PEG 8000 then re-clarifying via centrifugation at 30,000× g, 45 min | Anion exchange chromatography column | Precipitation with 10% PEG 8000 then size elution chromatography to obtain peak fraction. | [95] |
Plasma membrane | P-type H″-ATPase [85] | Arabidopsis seedlings | Homogenizing medium | 2770× g, 10 min, take supernatant; ultracentrifugation at 231,000× g, 35 min | Pellet | - | Multiple ultracentrifugation | Resuspend the pellet in PM-suspension medium | [96] |
Peroxisome | Catalase [97] | Arabidopsis rosette leaves | Grinding buffer | 5000× g, 1 min | Supernatant free from chloroplast and nuclei | 15–38% (v/v) Percoll gradient; centrifuge at 13,000× g, 12 min | 36% sucrose centrifuge at 39,000× g for 30 min | Leaf peroxisome fraction located at the bottom | [98] |
Autophagosome | Autophagy-related protein 8 (ATG8) [99] | Tobacco BY-2 cell suspension culture | Lysis buffer | 17,000× g, 5 min | Pellet | 30% Percoll; centrifuge at 50,000× g, 1 h | Place density marker beads on 30% Percoll solution and centrifuge again | Fractionate into 30 fractions | [100] |
Ribosome | Ribosomal Protein S6 (RPS6) [101] | Arabidopsis seedlings/leaves | Ribosome extraction buffer | 10,000× g, 15 min | Supernatant | Sucrose cushion, 149,000× g, 18 h | Resuspend ribosomal pellet in Staehelin A buffer; spin at 14,000× g for 15 min | Collect the supernatant | [102] |
Extracellular vesicles | Tetraspanin 8 [103] Syntaxin PENETRATION1 (PEN1) [104] | Arabidopsis rosettes | Vesicle isolation buffer (VIB) | 700× g, 20 min at 2 °C | Supernatant | - | Centrifuge successively at 10,000× g for 60 min, 40,000× g for 60 min, and 100,000× g for 60 min and obtain the pellet each time | Pellet resuspended in VIB | [104] |
Buffers | Components | Reference |
---|---|---|
Homogenization buffer one | 50 mM Tris–HCl (pH 7.4), 15 mM MgCl2, 10 mM KCl, 20% glycerol, 0.1% β-mercaptoethanol, 5 μg/mL leupeptin, and 2 μg/mL aprotinin. | [107] |
Nuclear extraction buffer | 10 mM MES (pH 5.2), 250 mM sorbitol, 10 mM NaCl, 5 mM NaF, 5 mM EDTA, 10 mM MgCl2, 0.024% Triton X-100, 0.1% bovine serum albumin, 1 mM fresh DTT, and Complete Protease Inhibitor Mixture. | [77] |
Enzyme buffer | 0.4 M Mannitol, 3.6 mM MES–KOH (pH 5.7), 2.0% (w/v) cellulase Onozuka RS, 0.5% (w/v) pectolyase Y-23, and 1.0% (w/v) Driselase. | [84] |
Protoplast solution | 1% (w/v) Cellulase Onozuka R10, 1% (w/v) Macerozyme R10, 0.4 M mannitol, 25 mM CaCl2, 5 mM mercaptoethanol, and 10 mM 2-morpholinoethanesulfonic acid (MES)-KOH (pH 5.7). | [87] |
Vacuole buffer | 0.45 M mannitol and 5 mM sodium phosphate 2 mM EDTA (pH 7.5). Keep on ice. The 200 mM sodium phosphate stock solution (pH 7.5) can be prepared by mixing 84 mL of 200 mM Na2HPO4 and 16 mL of 200 mM NaH2PO4. | [87] |
Enzyme mixture | 1.5% (w/v) cellulase R-10, 0.5% (w/v) macerozyme R-10, 5 mM 2-morpholinoethanesulfonic acid (MES), 0.1% (w/v) BSA, 10 mM CaCl2, and 0.4 M mannitol (pH 5.8). | [83] |
Chloroplast resuspension buffer | 0.3 M sorbitol, 20 mM Tricine-KOH (pH 7.6), 5 mM MgCl2, 2.5 mM EDTA. | [83] |
Homogenization buffer two | 500 mM sucrose, 10 mM KCl, 1 mM EDTA, 1 mM MgCl2 2 mM dithiothreitol (DTT), 0.1 mM phenylmethyl-sulfonyl fluoride (PMSF), and 150 mM Tricine-KOH pH 7.5. | [88] |
Extraction buffer for mitochondria | 0.4 M sorbitol, 0.2 M MOPS-Tris (pH 7.8), 7.5 mM EDTA, 1.5% (w/v) PVP-40, 0.1% [w/v] bovine serum albumin, and 2 mM DTT. | [90] |
Washing buffer | 0.33 M sorbitol and 50 mM MOPS-Tris (pH 7.5). | [90] |
Extraction buffer for Golgi membranes | 50 mM HEPES–KOH (pH 6.8), 0.4 M sucrose, 1 mM dithiothreitol (DTT), 5 mM MnCl2, and 5 mM MgCl2. | [92] |
Extraction buffer for Proteasomes | 50 mM potassium phosphate (pH 6), 2 mM MgCl2, 5% (v/v) glycerol, and 5 mM 2-mercaptoethanol supplemented with 10 mM ATP, 5% polyvinylpyrrolidone, 0.6% sodium metabisulfite, and 2 mM phenylmethylsulfonyl fluoride. 0.8% plant protease inhibitor mixture is added just before use. | [95] |
Homogenizing medium | 0.5 M sorbitol, 50 mM MOPS–KOH (pH 7.6), 5 mM EGTA, 5 mM EDTA, 1.5% (w/v) polyvinylpyrrolidone 40 (PVP-40, molecular weight 40,000), 0.5% (w/v) defatted-BSA, 2 mM phenylmethanesulfonyl fluoride (PMSF), 4 mM salicylhydroxamic acid (SHAM), and 2.5 mM 1,4-dithiothreitol (DTT). | [96] |
Plasma membrane (PM)-suspension medium | 10 mM MOPSKOH (pH 7.3), 1 mM EGTA, 0.3 M sucrose, 1 mM DTT. Store the stock solution without DTT at 4 °C. | [96] |
Grinding Buffer | 170 mM Tricine-KOH (pH 7.5), 1.0 M sucrose, 2 mM EDTA, 1% (w/v) BSA, 10 mM KCl, 1 mM MgCl2, 0.5% (w/v) PVP-40, and 5 mM DTT. | [98] |
Lysis buffer | 50 mM HEPES–KOH (pH 7.5) buffer containing 1 mM EDTA, 10 μM leupeptin, 10 μM pepstatin A, 1 mM AEBSF, and 0.4 M sorbitol. Mix 50 mL of 0.1 M HEPES–KOH (pH 7.5); 20 mL of 2 M sorbitol; 29 mL of water; and 1 mL of 0.1 M EDTA–NaOH (pH 8.0). Store at 4 °C. Take 10–20 mL of lysis buffer and add 1/100 volume of 1 mM leupeptin, 1/100 volume of 1 mM pepstatin A, and 1/100 volume of 0.1 M AEBSF immediately before use. | [100] |
Staehelin A buffer | 20 mM Tris–HCl (pH 7.5), 5 mM MgCl2, 1 mM sodium molybdate, and 1 mM dithiothreitol. | [102] |
Ribosome extraction buffer | 200 mM Tris–HCl (pH 7.5), 200 mM KCl, 25 mM EGTA, 36 mM MgCl2, 1 mM sodium molybdate, 1 mM dithiothreitol, 50 μg/mL cycloheximide, 50 μg/mL chloramphenicol, 80 mM β-glycerophosphate, 1% (v/v) Triton X-100, 1% (v/v) Brij 35, 1% (v/v) Tween 40, and 1% (v/v) NP40. | [102] |
Vesicle isolation buffer VIB | 20 mM MES, 2 mM CaCl2, and 0.1 M NaCl (pH 6). | [104] |
8. Experimental Challenges of Cell Fractionation
9. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Gomaa, A.E.; El Mounadi, K.; Parperides, E.; Garcia-Ruiz, H. Cell Fractionation and the Identification of Host Proteins Involved in Plant–Virus Interactions. Pathogens 2024, 13, 53. https://doi.org/10.3390/pathogens13010053
Gomaa AE, El Mounadi K, Parperides E, Garcia-Ruiz H. Cell Fractionation and the Identification of Host Proteins Involved in Plant–Virus Interactions. Pathogens. 2024; 13(1):53. https://doi.org/10.3390/pathogens13010053
Chicago/Turabian StyleGomaa, Amany E., Kaoutar El Mounadi, Eric Parperides, and Hernan Garcia-Ruiz. 2024. "Cell Fractionation and the Identification of Host Proteins Involved in Plant–Virus Interactions" Pathogens 13, no. 1: 53. https://doi.org/10.3390/pathogens13010053