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Root Gall Formation, Resting Spore Isolation and High Molecular Weight DNA Extraction of Plasmodiophora brassicae

Sara Mehrabi* Sara Mehrabi*
SS Suzana Stjelja*
CD Christina Dixelius

*Contributed equally to this work Published: Jun 5, 2018 DOI: 10.21769/BioProtoc.2864 Views: 6338

Edited by: Samik Bhattacharya

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Abstract

Isolation of DNA from obligate biotrophic soil-borne plant pathogens is challenging. This is because of their strict requirement of living plant tissue for their growth and propagation. A soil habitat further imposes risk of contamination from other microorganisms living in close vicinity of the plant roots. Here we present a protocol on how to prepare DNA suitable for advanced molecular analysis on the soil-borne pathogen Plasmodiophora brassicae, a peculiar unicellular plant pathogenic organism, causing disease on Crucifers. First, it is important to grow Brassica or Arabidopsis plants in infested soils below a temperature of 25 °C under moist conditions to promote root gall formation. Root galls should be harvested ahead of initiation of the decomposing process, no later than four or nine weeks post inoculation of Arabidopsis or Brassica plants, respectively. Resting spores with reduced numbers of soil organisms are achieved by gradient centrifugations of homogenized gall tissues. Treatments with 70% alcohol and a suit of different antibiotics promote P. brassicae purity. A CTAB-based procedure allows isolation of high quality DNA suitable for massive parallel sequencing analysis.

Keywords: Arabidopsis search Brassica search Clubroot search DNA search Plasmodiophora brassicae search Resting spores search Rhizaria search

Background

Plasmodiophora brassicae is a soil-borne plant pathogen causing root galls (clubs) in the Brassicaceae family including Arabidopsis. The clubroot disease has a major impact on oilseed rape (canola) and cabbage cultivation worldwide. P. brassicae is an obligate biotroph (require a host for growth) assigned to the supergroup Rhizaria, one of the least studied organism groups of eukaryotes (Sierra et al., 2016; Sibbald and Archibald, 2017). Phylogenetically, P. brassicae belongs to a plant pathogenic group of protists in Phytomyxea (Neuhauser et al., 2011 and 2014; Adl et al., 2012). Few genomes of related species are available, a circumstance which has considerably delayed the molecular analysis and genome comparisons. P. brassicae forms hardy resting spores in the clubs, spores that have the capacity to remain dormant for decades in the soil, ready for new rounds of root infections if a host plant grow nearby. Here we describe how to generate diseased plants, isolate resting spores from root galls followed by extraction of large amounts of DNA. This protocol is a further improvement and clarification of the procedures described in Schwelm et al. (2015). The outlined work is substantial but yields high-quality DNA suitable for long-read massive parallel sequencing.

Materials and Reagents

  1. Materials
    1. Safety glasses, gloves and lab coat
    2. Filter paper
    3. Plant pots, small pots (6 x 6 x 5 cm) and big pots (13 x 13 x 13 cm)
    4. Plant trays (34 x 22 x 4 cm)
    5. Soil (S-soil, Hasselfors Garden, Örebro, pH 5.5-6.5) composed of sighted light peat, black peat, perlite, sand, and lime
    6. Petri dishes 10 cm (ø)
    7. Miracloth Calbiochem® (Merck, catalog number: 475855 )
    8. Tubes (Falcon tube 15 ml, SARSTEDT, catalog number: 62.554.001 ; Falcon tube 50 ml, SARSTEDT, catalog number: 62.547.004 ; Eppendorf micro-tube 2 ml, SARSTEDT, catalog number: 72.695.500 )
    9. Plastic and glass beakers
    10. Scalpel
    11. Mortar, pestle and spoon (sterile and pre-chilled)
    12. Filtropur S0.2 (SARSTEDT, catalog number: 83.1826.001 )
  2. Plants
    1. Brassica rapa cv. ‘Granaat’ (European Clubroot Differential Set ECD-05)
    2. Arabidopsis thaliana Col-0

  3. Plasmodiophorid
    Plasmodiophora brassicae strain e3 (Fähling et al., 2004; the strain is available upon request to the authors)
    Note: Not all strains incite disease on Arabidopsis.
  4. Molecular biology working kit
    SpectrumTM Plant Total RNA Kit (Sigma-Aldrich, catalog number: STRN50 )
  5. Other reagents
    Note: *Those solutions are prepared with sterile distilled water.
    Spore isolation
    1. Ethanol (70%, 500 ml*)
    2. Sodium hypochlorite (1%, 500 ml*) (Commercial bleach)
    3. Sterile distilled water 5 x 1L
    4. Ficoll PM 400 (16% and 32%*) (Sigma-Aldrich, catalog number: F4375 )
    5. Rifampicin (100 mg/ml stock, prepared with methanol solvent) (Duchefa Biochemie, catalog number: R0146 )
    6. Streptomycin sulfate (100 mg/ml stock*) (Thermo Fisher Scientific, catalog number: 11860038 )
    7. Carbenicillin disodium (100 mg/ml stock, prepared with water solvent) (Duchefa Biochemie, catalog number: C0109 )
    8. Pimaricin (100 mg/ml stock*) (Merck, Sigma-Aldrich, catalog number: 1.07360 )
    9. Cefotaxime sodium (100 mg/ml stock*) (Duchefa Biochemie, catalog number: C0111 )
    10. Hygromycin B (50 mg/ml stock) (Duchefa Biochemie, catalog number: H0192 )
    11. Lysozyme from chicken egg white (4 mg/ml*) (Sigma-Aldrich, catalog number: L6876 )
    12. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 31434 )
    13. Potassium chloride (KCl) (Merck, catalog number: 104936 )
    14. di-Sodium hydrogen phosphate dihydrate (Na2HPO4) (Merck, catalog number: 119753 )
    15. Potassium dihydrogen phosphate (KH2PO4) (Merck, catalog number: 104873 )
    16. Tris-HCl (Trizma® hydrochloride solution) (1 M, pH 7.5) (Sigma-Aldrich, catalog number: T2694 )
    17. DNase I, RNase-free (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EN0521 )
    18. Proteinase K (20 mg/ml stock*) (Sigma-Aldrich, catalog number: RPROTK-RO )
    19. EDTA (0.5 M) (VWR, catalog number: 20294.294 )
    20. N-lauroylsarcosine sodium salt solution (1%, v/v) (Sigma-Aldrich, catalog number: 61747 )
    21. 1x PBS buffer (see Recipes)
    22. 1x TE buffer (pH 7.5) (see Recipes)
    23. Termination buffer (see Recipes)

    DNA extraction
    1. Liquid nitrogen
    2. Tris-HCl (Trizma® hydrochloride solution) (1 M, pH 7.5) (Sigma-Aldrich, catalog number: T2694 )
    3. EDTA (VWR, catalog number: BDH9232 )
    4. Sodium chloride (NaCl) (5 M stock *) (Sigma-Aldrich, catalog number: 31434 )
    5. Hexadecyltrimethylammonium bromide (CTAB) (Sigma-Aldrich, catalog number: H6269 )
    6. 2-Mercaptoethanol (VWR, catalog number: 436022A )
    7. Phenol:chloroform:isoamyl alcohol 25:24:1 (pH 7.5-8.0) (Carl Roth, catalog number: A156.2 )
    8. RNAse A, DNase and protease-free (10 mg/ml) (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: EN0531 )
    9. Chloroform, EMSURE® ACS, ISO, Reag. Ph. (Merck, catalog number: 1.02445.1000 )
    10. Ethanol (95%)
      Note: Pre-chill at -20 °C before use.
    11. Ethanol (70%)*
    12. 0.1x TE buffer (see Recipes)
    13. CTAB extraction buffer (see Recipes)
  6. Media and buffers (see Recipes)
    1. 1x PBS buffer
    2. 1x TE buffer
    3. Termination buffer
    4. CTAB extraction buffer

Equipment

  1. Growth chamber (Percival AR82L2/Split) or greenhouse
  2. Analytic balance (Mettler Toledo, model: AE100 )
  3. Household mixer (Rusta, catalog number: 90951442 , Max power 170 W)
  4. Liquid nitrogen container
  5. Water bath (JULABO, model: Julabo TW12, catalog number: 9550112 )
  6. pH meter (Mettler Toledo, model: SevenCompact S220, catalog number: 30019028 )
  7. Tabletop centrifuge for Eppendorf tubes (Thermo Fisher Scientific, Thermo ScientificTM, model: HeraeusTM FrescoTM 17 , catalog number: 75002420)
  8. Tabletop centrifuge for Falcon-tubes (Eppendorf, model: 5804/5804 R , catalog number: 5805000327)
  9. NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA)
  10. Microscope (Zeiss, Axioplan; camera: Leica Microsystems, model: Leica DFC295 )
  11. Stereoscope (Lecia, model: MZ FL III )
  12. Autoclave

Procedure

Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.

Category

Plant Science > Plant physiology > Biotic stress

Microbiology > Microbe-host interactions > Fungus

Molecular Biology > DNA > DNA extraction

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