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The Application of Quercetin to Study the Effect of Hsp70 Silencing on Plant Virus Infection in Nicotiana benthamiana Plants

Matthaios M. Mathioudakis Matthaios M. Mathioudakis
Ioannis Livieratos Ioannis Livieratos

Published: Vol 5, Iss 23, Dec 5, 2015 DOI: 10.21769/BioProtoc.1675 Views: 8301

Edited by: Zhaohui Liu

Original research article

The authors used this protocol in:

http://www.ncbi.nlm.nih.gov/pubmed/25162316

Dec 2014

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Abstract

Pepino mosaic virus (PepMV) is a mechanically-transmitted pathogen affecting tomato plants worldwide. Like with other plant viruses (Verchot, 2012), the heat shock cognate protein 70 homolog (Hsc70) was identified as an interactor of the PepMV coat protein (CP) (Mathioudakis et al., 2012). Here, we describe a pharmacological approach to silence Hsp70 in plants using quercetin (Mathioudakis et al., 2014), an Hsp70 protein expression flavonoid inhibitor (Hosokawa et al., 1990; Manwell and Heikkila 2007). In the case of Hsp70, this methodology represents a faster and easier approach than silencing of Hsp70 by reverse genetics assays, such as VIGS methodology. Fully expanded leaves of 2 to 3 weeks old Nicotiana benthamiana plants were infiltrated, using a syringe, with either quercetin (dissolved in DMSO) or DMSO (control plants). The plants were mechanically inoculated with PepMV virus inocula. The accumulation of Hsp70 and PepMV were analyzed on local leaves by immunoblot analysis 4 days post inoculation.

Materials and Reagents

  1. 1 ml syringe without a needle (BD, Nipro, catalog number: SY3 1 C100U ET )
  2. Mortar and pestles (Carl Roth GmbH + Co., catalog number: XL96.1 and XP01.1 )
  3. Pelet pestles (Nippon Genetics, catalog number: NG006 )
  4. 13 ml round base tubes (SARSTEDT AG & Co, catalog number: 62.515.006 )
  5. 1.5 ml microtubes (SARSTEDT AG & Co, catalog number: 72.690.001 )
  6. Nicotiana benthamiana leaves from 2 to 3 weeks old seedlings
  7. PepMV-Sp13 isolate infected material
  8. Quercetin hydrate (Sigma-Aldrich, catalog number: 337951 )
  9. Dimethyl sulfoxide (DMSO) (AppliChem GmbH, catalog number: A3009 )
  10. 10 mM Sodium carbonate (Na2CO3) (Sigma-Aldrich, Riedel-de Haen, catalog number: 31432 )
  11. Sodium dihydrogen phosphate monohydrate (0.5 M, pH 7.0) (AppliChem GmbH, catalog number: A3559 )
  12. Sodium phosphate dibasic (Na2HPO4) (Sigma-Aldrich, catalog number: S0876 )
  13. Potassium dihydrogen phosphate (KH2PO4) (Merck Millipore Corporation, catalog number: 104873 )
  14. Potassium chloride (KCl) (Sigma-Aldrich, Riedel-de Haen, catalog number: 31248 )
  15. Sodium Chloride (NaCl) (AppliChem GmbH, catalog number: A1149 )
  16. Magnesium Chloride 6-hydrate (MgCl2) (AppliChem GmbH, catalog number: A4425 )
  17. Silicon carbide (carborundum) 400 mesh (Sigma-Aldrich, catalog number: 357391 )
  18. Bromophenol blue (Sigma-Aldrich, catalog number: B-8026 )
  19. Coomassie Brilliant Blue (AppliChem GmbH, catalog number: A1092 )
  20. Glycerol (Sigma-Aldrich, catalog number: G6279 )
  21. Tris ultrapure (1.5 M, pH 8.8; 1 M, pH 6.8) (AppliChem GmbH, catalog number: A1086 )
  22. β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
  23. 30% 37.5:1 Acrylamide/Bis solution (Bio-Rad Laboratories, AbD Serotec®, catalog number: 161-0158 )
  24. 10% Ammonium persulfate (APS) (AppliChem GmbH, catalog number: A2941 )
  25. N, N, N´, N´-tetramethylethylenediamine (TEMED) (AppliChem GmbH, catalog number: A1148 )
  26. 10% Sodium dodecyl sulfate (SDS) (AppliChem GmbH, catalog number: A2263 )
  27. Glycine (AppliChem GmbH, catalog number: A1067 )
  28. Phenylmethanesulfonyl fluoride (PMSF) (Sigma-Aldrich, catalog number: P7626 )
  29. Methanol (Thermo Fisher Scientific, catalog number: M/4000/17 )
  30. Acetic acid (Sigma-Aldrich, catalog number: 33209 )
  31. PepMV CP polyclonal antibody (Neogen/Adgen Phytodiagnostics, catalog number: 1127-01 )
  32. HSP70 monoclonal antibody (Enzo Life Sciences, Stressgen, catalog number: N27F3-4 )
  33. Anti-rabbit IgG, Alkaline phosphatase-conjugated antibody (Promega Corporation, catalog number: S3731 )
  34. Anti-mouse IgG, Alkaline phosphatase-conjugated antibody (Promega Corporation, catalog number: S3721 )
  35. BCIP/NBP Color Development Substrate (Promega Corporation, catalog number: S3771 )
  36. PVDF membrane, Westran Clear Signal (Thermo Fisher Scientific, Whatmann, catalog number: 10485289 )
  37. Tween-20 (Sigma-Aldrich, catalog number: P2287 )
  38. Phosphate buffer saline (PBS) (see Recipes)
  39. Protein extraction buffer (see Recipes)
  40. 4x Laemmli buffer (see Recipes)
  41. SDS-PAGE buffers: Separation and Stacking gel buffers (see Recipes)
  42. Western Blot buffers: Running, Transfer, Wash and Detection buffers (see Recipes)

Equipment

  1. Plant growth chamber (25 °C, light:dark =16:8 h)
  2. Biofuge stratos highspeed table centrifuge (Thermo Fisher Scientific, Heraeus, catalog number: 75005282 )
  3. Fixed angle rotor #3334 (Thermo Fisher Scientific, Heraeus, catalog number: 75003334 )
  4. Fixed angle microliter rotor #3332 (Thermo Fisher Scientific, Heraeus, catalog number: 75003332 )
  5. Mini-PROTEAN 3 Cell Electrophoresis System (Bio-Rad Laboratories, AbD Serotec®, catalog number: 165-3301 )
  6. Mini Trans-Blot Electrophoretic Transfer Cell apparatus (Bio-Rad Laboratories, AbD Serotec®, catalog number: 170-3930 )
  7. Power Pack Supply model 200/2.0 (Bio-Rad Laboratories, AbD Serotec®, catalog number: 165-4761 )
  8. Western blot membranes were visualized using the Gel DocTM XR Molecular Imager & System (Bio-Rad Laboratories, AbD Serotec®, catalog number: 170-8195 EDU )
  9. Julabo Water-bath TW12 (Sigma-Aldrich, catalog number: Z615498 )
    Note: Pricing & availability is not currently available.

Software

  1. Quantification of the band intensity as absorbance units was conducted by Quantity One analysis software (Bio-Rad Laboratories, AbD Serotec®, catalog number: 170-9600)

Procedure

Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.

Category

Microbiology > Microbe-host interactions > Virus

Plant Science > Plant immunity > Host-microbe interactions

Plant Science > Plant molecular biology > Protein

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