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Establishing a Symbiotic Interface between Cultured Ectomycorrhizal Fungi and Plants to Follow Fungal Phosphate Metabolism
在培养的外生菌根真菌和植物之间建立共生界面以示踪真菌磷酸盐代谢   

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Plant, Cell & Environment
Feb 2017

Abstract

In ectomycorrhizal plants, the fungal cells colonize the roots of their host plant to create new organs called ectomycorrhizae. In these new organs, the fungal cells colonize the walls of the cortical cells, bathing in the same apoplasm as the plant cells in a space named the ‘Hartig net’, where exchanges between the two partners take place. Finally, the efficiency of ectomycorrhizal fungi to improve the phosphorus nutrition of their host plants will depend on the regulation of phosphate transfer from the fungal cells to plant cells in the Hartig net through as yet unknown mechanisms. In order to investigate these mechanisms, we developed an in vitro experimental device mimicking the common apoplasm of the ectomycorrhizae (the Hartig net) to study the phosphorus metabolism in the ectomycorrhizal fungus Hebeloma cylindrosporum when the fungal cells are associated or not with the plant cells of the host plant Pinus pinaster. This device can be used to monitor 32Phosphate efflux from the fungus previously incubated with 32P-orthophosphate.

Keywords: In vitro symbiotic interface (体外共生界面), 32Phosphate efflux measurement (32磷酸盐流出测定), Hebeloma cylindrosporum (粘花菇), Pinus pinaster (海岸松), Ectomycorrhizal symbiosis (外生菌根共生)

Background

The association between mycorrhizal fungi and plants is known to improve plant P nutrition (reviewed by Smith and Read, 2008; Plassard and Dell, 2010; Cairney, 2011; Smith et al., 2015). This positive effect is due to P uptake and P transport through the fungal cells exploring soil far away from the roots. The capacity of the fungus to take up P from the soil solution and to release P to mycorrhizal roots is therefore an important feature for its positive effect on plant P nutrition. In ectomycorrhizal symbiosis, we know (i) that the exchanges between the fungus and the plant occur in the Hartig net, located inside the ectomycorrhizae, and (ii) that there is no direct cellular connection via, for example plasmodesmata, between the plasma membrane of the fungal and the plant cells. Therefore, these exchanges are very difficult to study as they occur in the apoplasmic space of the Hartic net. Here, we describe an in vitro system enabling us to mimick this apoplasmic space for the ectomycorrhizal fungus Hebeloma cylindrosporum incubated with its host plant Pinus pinaster (Torres-Aquino et al., 2017). This method could be used with other fungal or plant species.

Materials and Reagents

  1. Sterile plastic 90 mm Petri dishes (Dominique DUTSCHER, Gosselin, catalog number: 688302 )
  2. Gloves, EcoSHIELD Natural Nitrile PF 250 (Dominique DUTSCHER)
  3. Multi-Purpose Silicone for kitchen or bathroom, 280 ml (Castorama, Rubson)
  4. PTFE (Polytetrafluoroethylene) microtube, 1.15 mm and 1.75 mm for internal and external diameter, respectively (Dominique DUTSCHER, PTFE, catalog number: 091932 )
  5. Filter paper without ash, grade 542, 185 mm diameter (Dominique DUTSCHER, WhatmanTM, catalog number: 1542185 )
  6. Autoclavable bags Polypropylene bag, 3 L, non-printed (Dominique DUTSCHER, catalog number: 140230 )
  7. Autoclave tape (Dominique DUTSCHER, catalog number: 490009 )
  8. Sterile 60 ml syringes 3 pieces (Dominique DUTSCHER, Omnifix, catalog number: 921010 )
  9. Sealing film for manual application, roll of 38 m x 100 mm (VWR, PARAFILM® M, catalog number: 291-1213 )
  10. Needles 18 G 0.9 x 40 mm (Dominique DUTSCHER, MicrolanceTM 3, catalog number: 301300 )
  11. Tubing, int diam 1.14 mm (Dominique DUTSCHER, Silicone, catalog number: 4906591 )
  12. Tubing, int diam 3.17 mm (Dominique DUTSCHER, Silicone, catalog number: 4906600 )
  13. 125 ml sterile polypropylene containers, red cap (Dominique DUTSCHER, catalog number: 688270 )
  14. Tips 200 µl for pipet (Dominique DUTSCHER, Sartorius, catalog number: 070468 )
  15. Tips 10 µl for pipet (Dominique DUTSCHER, Sartorius, catalog number: 077179 )
  16. Home-made needle holder for aeration
  17. Sterile syringe filters for air, 0.2 µm, 6.4 cm diam (Labomoderne, Midisart, catalog number: RS3320 )
  18. Nichrome wire, stainless steel, round, 22 gauge, 0.64 mm diameter (suppliers for electronic cigarettes)
  19. Valve luer polycarbonate one way (Cole-Parmer, catalog number: EW-30600-01 )
  20. Folding skirted caps, 14.9 mm diam (Dominique DUTSCHER, catalog number: 110603 )
  21. Paper for sterilisation (Dominique DUTSCHER, catalog number: 006950 )
  22. Pinus pinaster (maritime pine), seeds (Vilmorin, catalog number: PPA301 massif landais)
  23. Hebeloma cylindrosporum (ectomycorrhizal basidiomycete) (laboratory’s own collection, available upon request)
  24. Agar-agar (Sigma-Aldrich, catalog number: A7002-500G )
  25. D-glucose (Sigma-Aldrich, catalog number: G8270-1KG )
  26. Concentrated (30%) hydrogen peroxide (H2O2) solution (Sigma-Aldrich, catalog number: 216763-500ML-M )
  27. Manganese(II) sulfate monohydrate (MnSO4·H2O) (Sigma-Aldrich, catalog number: M7899-500G )
  28. Zinc sulfate heptahydrate (ZnSO4·7H2O) (Sigma-Aldrich, catalog number: Z0251-100G )
  29. Boric acid (H3BO3) (Sigma-Aldrich, catalog number: B6768-500G )
  30. Copper(II) sulfate pentahydrate (CuSO4·5H2O) (Sigma-Aldrich, catalog number: C8027-500G )
  31. Calcium nitrate tetrahydrate (Ca(NO3)2·4H2O) (Sigma-Aldrich, catalog number: C2786-500G )
  32. Potassium nitrate (KNO3) (Sigma-Aldrich, catalog number: P8291-500G )
  33. Potassium dihydrogen phosphate (KH2PO4) (EMD Millipore, catalog number: 105108 )
  34. Magnesium sulfate heptahydrate (MgSO4·7H2O) (Sigma-Aldrich, catalog number: 63138-250G )
  35. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9333-500G )
  36. Calcium chloride dihydrate (CaCl2·2H2O) (Sigma-Aldrich, catalog number: C5080-500G )
  37. Calcium hydroxide (Ca(OH)2) (EMD Millipore, catalog number: 102047 )
  38. Ammonium iron(III) citrate (NH4FeC6H5O7) (Sigma-Aldrich, catalog number: RES20400-A702X )
  39. Calcium sulfate dihydrate (CaSO4·2H2O) (EMD Millipore, catalog number: 102161 )
  40. 2-N-morpholino-ethanesulfonic acid, 4-morpholineethanesulfonic acid monohydrate (MES) (Sigma-Aldrich, catalog number: 69892-500G )
  41. Tris(hydroxymethyl)aminomethane (TRIS) (Sigma-Aldrich, catalog number: T1378-500G )
  42. 1 N sulfuric acid solution (EMD Millipore, catalog number: 1.09072.1000 )
  43. Agar plates for germination (see Recipes)
  44. 0.1 N Ca(OH)2 solution(see Recipes)
  45. Trace elements (1,000 ml) (see Recipes)
  46. Mineral salt base solutions (100 ml) (see Recipes)
  47. N1 + P solution (1,000 ml) (see Recipes)
  48. CaSO4 solution (0.2 mM) (see Recipes)
  49. Interaction medium (1,000 ml) (see Recipes)

Equipment

  1. A pair of stainless steel straight tweezers Wironit, Brucelles type, 130 mm (Dominique DUTSCHER, catalog number: 491037 )
  2. Glass bottles, 250 ml, ISO borosilicate, graduated (Dominique DUTSCHER, catalog number: 046413B )
  3. Glass bottles, 1000 ml, ISO borosilicate, graduated (Dominique DUTSCHER, catalog number: 046415 )
  4. Glass bottles, 2,000 ml, ISO borosilicate, graduated (Dominique DUTSCHER, catalog number: 046416 )
  5. Borosilicate glass funnel, 60° angle, 8 ml (Dominique DUTSCHER, catalog number: 068957 )
  6. A standalone burner (Dominique DUTSCHER, catalog number: 071109 )
  7. Butane gas cartridge for the burner (Dominique DUTSCHER, catalog number: 060415 )
  8. Automatic Piezo electronic gas lighter (Dominique DUTSCHER, catalog number: 076275 )
  9. Air-pumps for aquarium (SuperFish, model: air-box Nr.4 )
  10. Graduated borosilicate glass beaker 400 ml (Dominique DUTSCHER, catalog number: 068939 )
  11. Graduated borosilicate glass beaker 1,000 ml (Dominique DUTSCHER, catalog number: 068942 )
  12. Polypropylene economical beaker 3,000 ml with moulded graduations (Dominique DUTSCHER, catalog number: 391134 )
  13. Soda-lime glass Petri dish 150 x 25 mm (Dominique DUTSCHER, catalog number: 068522 )
  14. Glass tubing, length 150 mm, ext diameter 8 mm (VWR, AR-Glas®, catalog number: SCOR1193467 )
  15. Test tube, borosilicate glass, height 150 mm, internal diameter 20 mm (VWR, Duran, catalog number: 212-1120 )
  16. Stainless steel racks for 6 x 4 tubes of 16-20 mm diameter (Dominique DUTSCHER, catalog number: 854061 )
  17. A nail and cutting pliers
  18. A glass cutting knife (Sigma-Aldrich, catalog number: Z136441 )
  19. One stainless steel spatula, 235 mm (Dominique DUTSCHER, catalog number: 001809 )
  20. Autoclave
  21. Laminar flow cabinet, horizontal (Dominique DUTSCHER, catalog number: 486084 )
  22. Growth cabinet with controlled light, temperature and humidity (Binder, model: KBF P 720 )

Software

  1. Microsoft Excel for calculations
  2. Statistica 7.1 (StatSoft Inc., Tulsa, OK, USA)

Procedure

  1. Preparation of equipment
    1. Test tubes for plant (based on those given in Plassard et al., 1994)
      1. Cut with a glass knife small pieces (around 5 cm long) from a glass tubing (ext diameter 8 mm, equipment #14 in the Equipment section), hold the cut piece with a pair of tweezers and polish each side over a flame.
      2. Glue one piece of cut glass tubing inside a test tube (equipment #15 in the Equipment section), with silicone paste as shown in Figure 1. Place the tubes on a rack and leave them to dry for 24 h.


        Figure 1. Scheme of test tube used to grow young Pinus pinaster seedlings

      3. Cut the PTFE microtube (material #4 in Materials and Reagents section) in pieces of 25 cm and 30 cm long. Place one piece of each length inside the glass tubing and fill the space between the glass and the microtube with silicone paste. The long microtube will be used to supply nutrient solution whereas the short one could be used to withdraw the solution or to supply aeration. Leave to dry for 24 h.
      4. Cut small pieces (20 mm) of silicone tubing (internal diameter 1.14 mm, material #11 from the Materials and Reagents section) and place it over the end of a PTFE microtube. Close the extremity of a 200 µl tip by passing it through a flame. After cooling, insert the burned extremity into the second opening of the silicone tubing to obtain a stopper to close the microtube.
      5. Cut pieces of filter paper Whatman of 34 x 125 mm and place one per tube.
      6. Add 10 ml of glucose solution in each tube. This volume is enough to saturate the filter paper.
      7. Place the rack with tubes in an autoclavable bag. Close it with tape and put another autoclavable bag. Close it with tape and sterilise the whole at 115 °C for 40 min. Repeat the sterilization after 48 h. Glucose will promote the germination of spores of unwanted saprophytic fungal species belonging to the genus Penicillium, for example, that will be killed by the second sterilization. The tubes are ready to receive a germinated seed.
    2. Home-made syringe holders (Figure 2)
      Cut the different pieces of wood according to the dimensions given in Figure 2. For example, to hold 6 syringes, the top piece is 37 cm long x 6 cm wide and the one underneath is 32 cm long x 6 cm wide. Adjust and maintain the two pieces together to drill the holes in a two-by-two alignment. Afterwards, assemble all the pieces together as shown in Figure 2.


      Figure 2. Homemade holders to support syringes for incubation of mycelia in interaction medium

    3. Preparation of connectors for aeration (Figure 3)
      First, take an 18 G needle with a pair of tweezers, heat its collar over a flame to soften the glue and pull it with another pair of clamps. After cooling, adjust a small piece of silicone tube (1.14 mm internal diameter) of about 3 cm long on the top of the needle. Take a skirt cap and stitch 6 needles into its top and close its bottom by a 1 ml pipette tip previously cut at its finest end. If necessary, add silicone paste to fragile places to strengthen the airproof of the system. To the cut end of the blue pipette tip, add a piece of silicone tube (3.17 mm internal diameter) about 20 cm long that will be plugged later into a sterile air filter placed between the air pump and the connector (see Figure 4). Finally, place the whole system [connector + PTFE tubes + large diameter silicone tube] in an autoclavable bag and sterilize it by autoclaving (115 °C, 40 min).


      Figure 3. Homemade connector to aerate syringes during the incubation of mycelia in interaction medium


      Figure 4. Device used to incubate the plants with the mycelia of the ectomycorrhizal fungus. A. Syringe containing the interaction medium; B. Polycarbonate valve; C. Collection vessel; D. Mycelium; E. Root system of the interacting plant; F. Nichrome wire bearing the mycelium; G. Glass funnel to minimize evaporation; H. Home-made connector for gathering 4 to 7 PTFE microtubes; I. PTFE microtube for individual aeration; J. Sterile filter; K. Air pump. Each syringe can contain up to three P. pinaster seedlings.

    4. Preparation of syringes for incubation of the fungus, with or without the plant:
      Remove the plunger of each syringe and discard it. Using a hot nail, drill a hole at the top of the syringe opposite to the graduations giving the volume. This hole will be used to place the microtube for aeration inside the syringe. Place all drilled syringes in an autoclavable bag and sterilize it by autoclaving (115 °C, 40 min).

  2. Seed disinfection and germination
    1. First, prepare germination plates by pouring 25 ml of Agar-agar + glucose (see Recipes) solution in 90 mm Petri dishes. Generally, 8 seeds are put in one Petri dish and this ratio is used to calculate the total number of Petri dishes to be prepared. In our work we use seeds of maritime pine (Pinus pinaster Soland in Ait.) of Médoc provenance, originating from Landes-Sore-VG (France). The seeds, whose number is converted in g using the weight of 10 seeds, are first soaked for 48 h at 4 °C in deionized water as we noticed that this soaking improved the success of disinfection.
    2. After removing water, the seeds are placed into a large Petri dish (diameter 150 mm) in a laminar flow cabinet. They are then covered with pure, concentrated H2O2 (30%). After 50 min, H2O2 solution is discarded. Then, the seeds are rinsed several times with roughly 1 L of sterile deionized water.
    3. After the last rinse, all the water is withdrawn from the dish with a sterile syringe and the seeds are allowed to become completely dry in the laminar flow cabinet. The seeds are then oriented with the root down and deposited on the solid medium, on a single line in the middle of the dish. The dish is closed with sealing film and placed vertically in a box to save space and to get straight roots that do not penetrate into the solid medium. The box is incubated in the dark, at 25 °C. The first seeds (about 10%) germinate after 10 days and the maximum of germination occurs within 2 to 3 weeks.

  3. Placement of germinated seeds in test tubes
    1. Open the bag containing sterile test tubes in a laminar flow cabinet. Discard the largest possible volume of glucose solution by pouring it into a sterile beaker. This step is very important to avoid contaminations.
    2. Then, add 15 ml of nutrient solution (N1 + P, see Recipes) in each tube of a rack with the 60 ml syringe. Cut the sealing film into strips of 25 x 150 mm (one per tube). Open the Petri dishes with germinated seeds by discarding the sealing film.
    3. Take a tube in one hand and move the filter paper away from the wall of the glass tube with a flame-sterilized spatula held in the other hand. Take a germinated seedling with tweezers and insert its root between the glass and the paper. Use the seeds with a root length of at least 7 cm and with teguments still covering the cotyledons. Gently press the filter paper against the root and close the tube with the sealing film by making several turns around the hypocotyl to completely close all the openings. Place the tube with the seedling (Figure 1) in another rack outside the laminar flow cabinet.

  4. Culture conditions of plants
    1. Place the tubes in a growth cabinet with controlled light and humidity. We generally use the following parameters to grow the plants: a 16/8 h light/dark cycle at 25 °C/20 °C, 60%/80% RH, CO2 concentration of about 350 x 10-6 dm-3 and a PAR (Photosynthetically Active Radiation) of approximately 400 μmol m-2 sec-1 (400-700 nm).
    2. Maintain the nutrient solution at 15 ml by adding regularly new nutrient solution under sterile conditions. Use a needle (18 G) equipped with a 40 mm-long piece of silicone tubing (int diam 1.14 mm). Several needles are prepared, placed in 125 ml polypropylene containers and autoclaved at 121 °C for 20 min.
    3. At the time of re-filling the test tubes, open the container under sterile conditions and connect a needle onto a sterile 60 ml Luer syringe filled with nutrient solution. Open first the PTFE microtube of a test tube by taking off the stopper (silicone tubing + burned 200 µl tip). Connect the open extremity of the microtube with the silicone tubing protruding from the syringe. Push the nutrient solution into the test tube until the wanted volume. Withdrawn the syringe and close back the PTFE microtube with its stopper.

  5. Plant preparation for monitoring the fate of phosphate
    1. In our work (Torres-Aquino et al., 2017) we used 2-month old maritime pines. Six days before the incubation with the mycelia, the nutrient solution of each test tube is replaced by 30 ml of sterile CaSO4 (see Recipes). Six or seven test tubes are connected together by plugging one of the PTFE microtube in a home-made needle holder (Figure 3).
    2. The holder is connected to a sterile filter and the racks with the plants are transferred into the growth cabinet. The filter is then connected to an air pump to provide a sterile aeration to each test tube. After 5 days, the CaSO4 solution is replaced by 30 ml of interaction medium (see Recipes) for 24 h to acclimatize the roots to this new medium.

  6. Incubation with the ectomycorrhizal fungus
    1. Disconnect the PTFE microtube from the connector and remove the sealing film. Take the plant out of the test tubes by hand. Add it to the 60 ml syringes containing a 32P labelled mycelium previously grown and rinsed as described in Becquer et al. (2017). In our work, we put three 2-month-old maritime pine seedlings in each syringe that contains 60 ml of interaction medium and a mycelium of the ectomycorrhizal basidiomycete Hebeloma cylindrosporum (about 0.2 g of fresh weight) suspended in the syringe by a nichrome wire (see Figure 4).
    2. Take a connector with PTFE microtubes (Figure 3) and insert one PTFE microtube into each syringe through the hole and connect the connector to an air pump. Adjust the flow rate of the pump and the position of the PTFE microtubes so as to obtain equivalent ventilation in all syringes. Add a glass funnel at the top of each syringe to minimize the evaporation of the medium. Incubate the plants and the fungi under laboratory light continuously for up to 48 h.
    3. To study the effect of plant on 32P efflux from the fungus, take 1 ml of interaction medium after 6, 24 and 48 h of incubation and measure the radioactivity as described in detail in our previous protocol (Becquer et al., 2017). Before sampling, do not forget to record the actual volume of interaction medium in the syringe. At the end of incubation, record the fresh weight of each mycelium and measure their radioactivity (for details, see Becquer et al., 2017). Take also each plant; blot it between filter paper sheets, separate roots from shoots and record their fresh weight. Then place the whole plant (roots + shoots) in a 20 ml scintillation vial and measure the radioactivity as described in Becquer et al. (2017).

  7. Calculations with 32P labelled mycelia
    1. Refer to our previous protocol to correct the raw data (generally given in cpm = counts per minute) for decay (Becquer et al., 2017).
    2. Calculate the net amount of radioactivity released by the fungus after 6, 24 and 48 h (or any other incubation period) using the following equation written in Excel:
      cpmt0-t6 = cpmt6 x [(60 + Vbs IMt6)/2]
      cpmt24-t6 = cpmt24 x [(Vas IMt6 + Vbs IMt24/2]
      cpmt24-t48 = cpmt48 x [(Vas IMt624 + Vbs IMt48)/2]
      with:
      cpmti= counts per minute measured in 1 ml of IM at time i
      Vas IMti = volume (ml) of interaction medium after sampling at time i
      Vbs IMti = volume (ml) of interaction medium before sampling at time i
      Use these data to calculate the amounts of radioactivity lost by the fungus over the different incubation periods:
      0-6 h = cpmt0-t6
      0-24 h = cpmt0-t6 + cpmt6-t24
      0-48 h = cpmt0-t6 + cpmt6-t24 + cpmt24-t48
    3. To calculate the whole amount of radioactivity lost by the fungus over 48 h, add the values of cpm measured in interaction medium (0-48 h) with those measured in plants.
    4. Add these previous values with those measured in the fungus before plant addition to get the total amount of radioactivity taken up by the fungus.
    5. Transform the values of cpm in Becquerel using the formula given in Becquer et al. (2017).

Data analysis

When the fungus is labelled with 32P (see Becquer et al., 2017), the number of replicates per treatment should be at least 6 and the experiments should be repeated twice. Results can be expressed either as the percentage of initial radioactivity lost by the fungus or per g of fungal fresh weight. The normality of data is tested using the Kolmogorov Smirnov test and, where necessary, the data are either square root or log10 transformed prior to analysis to meet the assumptions of ANOVA. The effect of incubation time (e.g., 6, 24 or 48 h) and of the biological conditions (the fungus alone versus the fungus with the plant) can be assessed using one way- or two way-ANOVA.

Notes

  1. In the disinfection step of maritime pine seeds, the person must wear gloves to avoid any burning from skin contact with pure, concentrated H2O2.
  2. The seeds must have a good contact with H2O2 for efficient disinfection.
  3. At the time of seedling transfer into the test tube, it is very important that they have their tegument covering the cotyledons. Without this tegument, the young seedling will dry very quickly and die.
  4. In case the tegument has been lost, the seedlings can be protected from air drying by covering with a filter paper, well-moistened with deionized water.
  5. Depending on the number of plants required for the experiments, it is possible to place 2 germinated seeds side by side in the tube. In this case, it is necessary to monitor the level of the nutrient solution very frequently.
  6. Since the risk of contamination of filter paper with bacteria or saprophytic fungi is never zero, we usually prepare 10% more plants than needed.

Recipes

  1. Agar plates for germination
    1. Prepare the glucose solution by dissolving 2 g of D-glucose in 1 L of deionized water
    2. Take two 1 L glass bottles, add 7.5 g of Agar-agar and pour 500 ml of glucose solution in each bottle
    3. Sterilize the agar medium at 121 °C for 20 min
    4. Pour the cooled medium (55-60 °C) in Petri dishes 90 mm diameter
  2. 0.1 M Ca(OH)2 solution
    1. Pour 100 ml of deionized water in a 250 ml-glass bottle
    2. Add 0.74 g of Ca(OH)2 and shake
    3. The powder may not completely dissolve and the solution must be shaken by hand to resuspend the powder before use
    4. Store at room temperature
  3. Trace elements (1,000 ml)
    3.08 g MnSO4·H2O
    4.41 g ZnSO4·7H2O
    2.82 g H3BO3
    0.98 g CuSO4·5H2O
    0.29 g Na2MoO4·2H2O
    Add deionized H2O to 1,000 ml, store at 4 °C
  4. Mineral salt base solutions (100 ml)


    All these solutions are stored at 4 °C
  5. N1 + P solution (1,000 ml)
    1. Add in a 1 L-beaker approximately 500 ml of deionized water and the following volumes of mineral base solutions:
      0.2 ml Ca(NO3)2
      0.6 ml KNO3
      0.2 ml KCl
      0.2 ml KH2PO4
      0.5 ml ferric ammonium citrate
    2. Add also trace elements 0.2 ml
    3. Complete the volume to 1 L and adjust the pH to 5.5
    4. Sterilize the solution at 121 °C for 20 min
  6. CaSO4 solution (0.2 mM)
    1. Add in a 1 L-beaker 1,000 ml of deionized water and 34.4 mg of calcium sulphate
    2. Shake the solution carefully and adjust the pH to 5.5 if necessary with 0.1 N H2SO4 or 0.1 M Ca(OH)2
    3. Sterilize the solution at 121 °C for 20 min
  7. Interaction medium (3,000 ml)
    1. Add in a 3 L-beaker approximately 1.5 L of deionized water and the following volumes of mineral base solutions:
      0.6 ml MgSO4
      1.5 ml CaCl2
      3.2 g of MES (final concentration of 5 mM)
      1.82 g of TRIS (final concentration of 5 mM)
    2. Complete to 3 L with deionized water and adjust the pH to 5.5 with 1 N H2SO4
    3. Pour 1.5 L of medium into 2 L glass bottles and sterilize by autoclaving (20 min, 121 °C)

Acknowledgments

This research was supported by INRA (France) through annual funding devoted to their researchers and a fellowship through a Contract for Young Scientist (CJS) granted to Adeline Becquer, and by CONACYT (Mexico) through a Ph-D fellowship granted to Margarita Torres-Aquino. The protocol is adapted from our previous work (Plassard et al., 1994; Torres-Aquino et al., 2017). We also thank the three anonymous reviewers for their helpful comments to improve the protocol.

References

  1. Becquer, A., Torres-Aquino, M., Le Guernevé, C., Amenc, L.K., Trives-segura, C., Staunton, S., Quiquampoix, H. and Plassard, C. (2017). A Method for Radioactive labelling of Hebeloma cylindrosporum to study plant-fungus interactions. Bio Protoc 7(20): e2576.
  2. Cairney, J. W. G. (2011). Ectomycorrhizal fungi: the symbiotic route to the root for phosphorus in forest soils. Plant Soil 344: 51-71.
  3. Plassard, C., Barry, D., Eltrop, L. and Mousain, D. (1994). Nitrate uptake in maritime pine (Pinus pinaster) and the ectomycorrhizal fungus Hebeloma cylindrosporum: effect of ectomycorrhizal symbiosis. Can J Bot 72: 189-197.
  4. Plassard, C. and Dell, B. (2010). Phosphorus nutrition of mycorrhizal trees. Tree Physiol 30(9): 1129-1139.
  5. Smith, S. E. and Read, D. J. (2008). Mycorrhizal symbiosis. 3rd edition. Academic Press.
  6. Smith, S. E., Anderson, I. C. and Smith, F. A. (2015). Mycorrhizal associations and phosphorus acquisition: from cells to ecosystems. Annual Plant Reviews 48: 409-440.
  7. Torres-Aquino, M., Becquer, A., Le Guerneve, C., Louche, J., Amenc, L. K., Staunton, S., Quiquampoix, H. and Plassard, C. (2017). The host plant Pinus pinaster exerts specific effects on phosphate efflux and polyphosphate metabolism of the ectomycorrhizal fungus Hebeloma cylindrosporum: a radiotracer, cytological staining and 31P NMR spectroscopy study. Plant Cell Environ 40(2): 190-202.

简介

在外生菌根植物中,真菌细胞定植于其宿主植物的根部,以产生称为外生菌根的新器官。在这些新器官中,真菌细胞定居在皮质细胞的壁上,与称为“Hartig网”的空间中的植物细胞在同一质粒中沐浴,两地之间的交流发生。最后,外源菌根真菌提高其宿主植物的磷营养的效率将取决于通过尚未知的机制调节从真菌细胞到Hartig网中植物细胞的磷酸转移。为了研究这些机制,我们开发了一种体外实验装置,模拟外生菌根(Hartig网)的常见质粒来研究外生菌根真菌中的磷代谢。 当真菌细胞与寄主植物“Pinus pinaster”的植物细胞相关联时。该装置可用于监测从先前与 32 P-正磷酸盐一起培养的真菌的磷酸盐外排。
【背景】已知菌根真菌和植物之间的关联可以改善植物P营养(由Smith和Read,2008; Plassard和Dell,2010; Cairney,2011; Smith等人,2015年)。这种积极作用是由于P摄取和P运输通过真菌细胞探索远离根的土壤。因此,真菌从土壤溶液中吸收P并将P释放到菌根的能力是其对植物P营养的积极影响的重要特征。在外生菌根共生中,我们知道(i)真菌和植物之间的交换发生在位于外生菌根内的Hartig网中,和(ii)在质膜之间没有直接的细胞连接通过例如plasmodesmata的真菌和植物细胞。因此,这些交流是非常困难的,因为它们发生在Hartic网络的质外界。在这里,我们描述了体外体系,使我们能够模仿这种外源菌根真菌的球形体空间与其主体植物Pinus pinaster 孵化的圆筒孢子(Hebeloma cylindrosporum) Torres-Aquino等人,2017)。该方法可用于其他真菌或植物物种。

关键字:体外共生界面, 32磷酸盐流出测定, 粘花菇, 海岸松, 外生菌根共生

材料和试剂

  1. 无菌塑料90毫米培养皿(Dominique DUTSCHER,Gosselin,目录号:688302)
  2. 手套,EcoSHIELD天然丁腈PF 250(Dominique DUTSCHER)
  3. 多功能硅胶厨房或浴室,280毫升(Castorama,Rubson)
  4. PTFE(聚四氟乙烯)微管,内径和外径分别为1.15 mm和1.75 mm(Dominique DUTSCHER,PTFE,目录号:091932)
  5. 不含灰分的滤纸,等级542,直径185毫米(Dominique DUTSCHER,Whatman TM,目录号:1542185)
  6. 高压灭菌袋聚丙烯袋,3L,未印刷(Dominique DUTSCHER,目录号:140230)
  7. 高压灭菌胶带(Dominique DUTSCHER,目录号:490009)
  8. 无菌60 ml注射器3件(Dominique DUTSCHER,Omnifix,目录号:921010)
  9. 用于手动应用的密封膜,38m×100mm的卷(VWR,PARAFILM M,目录号:291-1213)
  10. 针18 G 0.9 x 40 mm(Dominique DUTSCHER,Microlance TM 3,目录号:301300)
  11. 管道,直径1.14 mm(Dominique DUTSCHER,Silicone,目录号:4906591)
  12. 管,直径3.17毫米(Dominique DUTSCHER,Silicone,目录号:4906600)
  13. 125毫升无菌聚丙烯容器,红帽(Dominique DUTSCHER,目录号:688270)
  14. 提示200μl用于移液器(Dominique DUTSCHER,Sartorius,目录号:070468)
  15. 提示10μl用于移液器(Dominique DUTSCHER,Sartorius,目录号:077179)
  16. 自制针架用于曝气
  17. 空气无菌注射器过滤器,0.2μm,6.4 cm直径(Labomoderne,Midisart,目录号:RS3320)
  18. 镍铬合金线,不锈钢,圆形,22号,直径0.64毫米(电子香烟供应商)
  19. 阀门鲁尔聚碳酸酯单向(Cole-Parmer,目录号:EW-30600-01)
  20. 折叠裙边帽,14.9毫米直径(Dominique DUTSCHER,目录号:110603)
  21. 灭菌纸(Dominique DUTSCHER,目录号:006950)
  22. 松属松属(Maritime pine),种子(Vilmorin,目录号:PPA301 massif landais)
  23. (octulum菌根菌纲)(ectomycorrhizal担子菌)(实验室自己的收集,可根据要求提供)
  24. 琼脂(Sigma-Aldrich,目录号:A7002-500G)
  25. D-葡萄糖(Sigma-Aldrich,目录号:G8270-1KG)
  26. 浓缩(30%)过氧化氢(H 2 O 2 O 2)溶液(Sigma-Aldrich,目录号:216763-500ML-M)
  27. 硫酸锰(II)一水合物(MnSO 4•H 2 O)(Sigma-Aldrich,目录号:M7899-500G)
  28. 硫酸锌七水合物(ZnSO 4•7H 2 O)(Sigma-Aldrich,目录号:Z0251-100G)
  29. 硼酸(H 3 3 BO 3)(Sigma-Aldrich,目录号:B6768-500G)
  30. 硫酸铜(II)五水合物(CuSO 4•5H 2 O)(Sigma-Aldrich,目录号:C8027-500G)
  31. 硝酸钙四水合物(Ca(NO 3 3)2•4H 2 O)(Sigma-Aldrich,目录号:C2786-500G)
  32. 硝酸钾(KNO 3)(Sigma-Aldrich,目录号:P8291-500G)
  33. 磷酸二氢钾(KH 2 PO 4)(EMD Millipore,目录号:105108)
  34. 硫酸镁七水合物(MgSO 4•7H 2 O)(Sigma-Aldrich,目录号:63138-250G)
  35. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9333-500G)
  36. 氯化钙二水合物(CaCl 2•2H 2 O)(Sigma-Aldrich,目录号:C5080-500G)
  37. 氢氧化钙(Ca(OH)2)(EMD Millipore,目录号:102047)
  38. 柠檬酸铵(III)(NH 4)FeCl 6 H 5 O 7(Sigma-Aldrich,目录号号码:RES20400-A702X)
  39. 硫酸钙二水合物(CaSO 4•2H 2 O)(EMD Millipore,目录号:102161)
  40. 2-N-吗啉代 - 乙磺酸,4-吗啉乙磺酸一水合物(MES)(Sigma-Aldrich,目录号:69892-500G)
  41. 三(羟甲基)氨基甲烷(TRIS)(Sigma-Aldrich,目录号:T1378-500G)
  42. 1N硫酸溶液(EMD Millipore,目录号:1.09072.1000)
  43. 用于发芽的琼脂板(参见食谱)
  44. 0.1 N Ca(OH)2溶液(参见食谱)
  45. 痕量元素(1,000毫升)(见食谱)
  46. 矿物盐溶液(100ml)(参见食谱)
  47. N1 + P溶液(1000毫升)(参见食谱)
  48. CaSO 4 溶液(0.2mM)(参见食谱)
  49. 相互作用介质(1,000毫升)(见配方)

设备

  1. 一双不锈钢直镊子Wironit,Brucelles型,130 mm(Dominique DUTSCHER,目录号:491037)
  2. 玻璃瓶,250毫升,ISO硼硅酸盐,刻度(Dominique DUTSCHER,目录号:046413B)
  3. 玻璃瓶,1000毫升,ISO硼硅酸盐,毕业(Dominique DUTSCHER,目录号:046415)
  4. 玻璃瓶,2000毫升,ISO硼硅酸盐,毕业(Dominique DUTSCHER,目录号:046416)
  5. 硼硅玻璃漏斗,60°角,8ml(Dominique DUTSCHER,目录号:068957)
  6. 独立燃烧器(Dominique DUTSCHER,目录号:071109)
  7. 用于燃烧器的丁烷气瓶(Dominique DUTSCHER,目录号:060415)
  8. 自动压电式气体打火机(Dominique DUTSCHER,目录号:076275)
  9. 水族馆气泵(SuperFish,型号:机箱Nr.4)
  10. 刻度硼硅酸盐玻璃烧杯400ml(Dominique DUTSCHER,目录号:068939)
  11. 刻度硼硅酸盐玻璃烧杯1000毫升(Dominique DUTSCHER,目录号:068942)
  12. 聚丙烯经济烧杯3000毫升带成型刻度(Dominique DUTSCHER,目录号:391134)
  13. 钠钙玻璃培养皿150 x 25毫米(Dominique DUTSCHER,目录号:068522)
  14. 玻璃管,长度150 mm,外径8 mm(VWR,AR-Glas ®,目录号:SCOR1193467)
  15. 试管,硼硅酸盐玻璃,高度150mm,内径20mm(VWR,Duran,目录号:212-1120)
  16. 直径16-20 mm的6 x 4管不锈钢机架(Dominique DUTSCHER,目录号:854061)
  17. 钉子和剪钳
  18. 玻璃切割刀(Sigma-Aldrich,目录号:Z136441)
  19. 一个不锈钢铲刀,235毫米(Dominique DUTSCHER,目录号:001809)
  20. 高压灭菌器
  21. 层流柜,水平(Dominique DUTSCHER,目录号:486084)
  22. 具有受控光,温湿度的生长柜(Binder,型号:KBF P 720)

软件

  1. 用于计算的Microsoft Excel
  2. Statistica 7.1(StatSoft Inc.,Tulsa,OK,USA)

程序

  1. 准备设备
    1. 用于植物的试管(根据Plassard et al。,1994)中给出的试管,
      1. 用玻璃刀切割小件(约5厘米长)从玻璃管(外径8毫米,设备部分的设备#14),用一对镊子握住切割片,并在每个火焰上擦亮。 br />
      2. 将一块切割的玻璃管粘在试管(设备部分的设备#15)内,用硅胶粘贴,如图1所示。将管放置在架子上,使其干燥24小时。


        图1.用于种植幼苗的试管方案幼苗

      3. 将PTFE微管(材料和试剂部分的材料#4)切成25厘米和30厘米长的碎片。将一段长度放在玻璃管内,并用硅胶填充玻璃和微管之间的空间。长的微管将用于提供营养液,而较短的微管可用于提取溶液或提供通气。离开24小时。
      4. 切割硅胶管(内径1.14 mm,材料和试剂部分的材料#11)的小件(20 mm),并将其放在PTFE微管的末端。通过穿过火焰关闭200μl尖端的末端。冷却后,将燃烧的末端插入硅胶管的第二个开口,以获得一个关闭微管的塞子。
      5. 切成34×125毫米的滤纸Whatman,每管放置一个。
      6. 在每个管中加入10ml葡萄糖溶液。该卷足以使滤纸饱和。
      7. 将管子放在高压灭菌袋中。用胶带将其关闭并放入另一个可高压灭菌的袋子。用胶带封闭,并在115°C下彻底灭菌40分钟。 48小时后重复灭菌。葡萄糖将促进属于青霉属的不想要的腐生真菌的孢子的发芽,例如将被第二次灭菌杀死的孢子。管子准备好接受发芽的种子。
    2. 自制注射器支架(图2)
      根据图2中给出的尺寸切割不同的木材。例如,要保持6个注射器,顶部的长度为37厘米长x 6厘米,下面的长度为32厘米长×6厘米宽。调整并保持两个部件在一起,以双向对准钻孔。之后,将所有的部分组合在一起,如图2所示

      图2.支持用于在相互作用介质中孵育菌丝体的注射器的自制支架

    3. 准备连接器曝气(图3)
      首先,用一双镊子取18针针,将其衣领加热一段火焰,软化胶水并用另一对夹子拉。冷却后,调整针头顶部约3厘米长的小片硅胶管(内径1.14毫米)。拿一个裙帽,将6针缝合到其顶部,并通过1毫升移液管尖端将其底部关闭,先前在其最好的端部切割。如有必要,可将硅胶加入脆弱处,以加强系统的防风。在蓝色移液管尖端的切割端,加入约20厘米长的硅胶管(3.17毫米内径),稍后将其塞入空气泵和连接器之间的无菌空气过滤器(见图4)。最后,将整个系统[连接器+ PTFE管+大直径硅胶管]置于高压灭菌袋中,并通过高压灭菌(115°C,40分钟)灭菌。


      图3.在相互作用介质中培养菌丝体期间充气注射器的自制连接器


      图4.用于将植物与外生菌根真菌菌丝体孵育的装置。 A.含有相互作用介质的注射器; B.聚碳酸酯阀;收集船D.菌丝体E.相互作用植物根系; F.镍铬合金丝带有菌丝体; G.玻璃漏斗以最小化蒸发; H.用于聚集4至7个PTFE微管的自制连接器; I.用于单独曝气的PTFE微管;无菌过滤器K.空气泵。每个注射器最多可以包含三个P。苗木幼苗。

    4. 准备用于温育真菌的注射器,有或没有植物:
      取出每个注射器的柱塞并将其丢弃。使用热指甲,在与注射量相对应的刻度表相对的注射器顶部钻一个孔。该孔将用于将微管放置在注射器内进行通气。将所有钻孔的注射器放在高压灭菌袋中,并通过高压灭菌(115°C,40分钟)灭菌。

  2. 种子消毒和发芽
    1. 首先,通过在90毫升培养皿中倒入25毫升琼脂+葡萄糖(参见食谱)溶液来制备萌发板。通常将8粒种子放在一个培养皿中,并使用该比例计算待制备的培养皿总数。在我们的工作中,我们使用起源于Landes-Sore-VG(法国)的Médoc来源的海上松木( Soland in Ait)的种子。我们注意到这种浸泡提高了消毒的成功率,使用10粒种子的重量将其数量转换成g的种子在4℃下在去离子水中浸泡48小时。
    2. 除去水后,将种子放入层流柜中的大培养皿(直径150mm)中。然后用纯的,浓缩的H 2 O 2 O 3(30%)覆盖它们。 50分钟后,丢弃H 2 O 2 O 2溶液。然后,用约1L无菌去离子水将种子冲洗数次。
    3. 在最后一次冲洗之后,用无菌注射器从盘中取出所有的水,并使种子在层流柜中变得完全干燥。然后将种子定向于根部,并将其沉积在固体培养基上,在盘的中间的单个线上。该盘用密封膜封闭,垂直放置在盒子中,以节省空间,并获得不渗入固体介质的直根。将箱子在黑暗中在25℃下孵育。第一批种子(10%左右)10天后发芽,2〜3周内发芽最大。

  3. 发芽种子放置在试管中
    1. 在层流柜中打开包含无菌试管的袋子。通过将葡萄糖溶液倒入无菌烧杯来丢弃最大体积的葡萄糖溶液。这一步对于避免污染非常重要。
    2. 然后,用60 ml注射器加入15 ml的营养液(N1 + P,参见食谱)。将密封膜切成25×150mm的条(每管一个)。通过丢弃密封膜打开具有发芽种子的培养皿。
    3. 用一只手拿管,将滤纸从玻璃管的墙壁上移开,另一只手握着灭火的抹刀。用镊子取出发芽的幼苗,将其根部插入玻璃和纸张之间。使用根长度至少为7厘米的种子,并且仍然覆盖子叶的牙膏。轻轻地将滤纸按压根部,并用密封膜封闭管子,沿着下胚轴绕几圈,完全关闭所有的开口。将管子与幼苗(图1)放置在层流柜外面的另一个机架中。

  4. 植物的培养条件
    1. 将管放在具有受控的光照和湿度的生长箱中。我们通常使用以下参数来生长植物:在25℃/ 20℃,60%/ 80%RH,CO 2浓度为约350×x 16℃的光/暗循环大约400μmol/平方米的PAR(光合有源辐射) -3 (400-700nm)。
    2. 通过在无菌条件下定期添加新的营养液,将营养液维持在15毫升。使用配有40毫米长的硅胶管(直径1.14毫米)的针(18 G)。制备几根针,置于125ml聚丙烯容器中,并在121℃高压灭菌20分钟。
    3. 在重新填充试管时,在无菌条件下打开容器,并将针连接到装有营养液的无菌60ml Luer注射器上。通过取下塞子(硅胶管+燃烧的200μl尖端)先打开试管的PTFE微管。将微型管的开放末端与从注射器突出的硅胶管连接。将营养液推入试管直到想要的体积。取出注射器并用其塞子关闭PTFE微管。

  5. 植物制备监测磷酸盐的命运
    1. 在我们的工作(Torres-Aquino等人,2017年)中,我们使用了2个月大的海洋松树。在与菌丝体孵育前6天,每个试管的营养液被30ml无菌CaSO 4(替代品)代替。六个或七个试管通过将PTFE微管中的一个插入自制针架中而连接在一起(图3)。
    2. 保持器连接到无菌过滤器,并且具有植物的架子被转移到生长箱中。然后将过滤器连接到空气泵以对每个试管提供无菌曝气。 5天后,用30ml的相互作用介质(参见食谱)将CaSO 4溶液置换24小时,以将根适应于该新培养基。

  6. 孵化与外生菌根真菌
    1. 从连接器上拆下PTFE微管并拆下密封膜。用手拿出试管。将其加入到60ml注射器中,该注射器含有以前生长并按照Becquer等人的描述冲洗的 32 P标记的菌丝体。 (2017年)。在我们的工作中,我们在每个注射器中放置三个2个月大的海洋松树苗,每个注射器中含有60毫升相互作用介质和一个放置在外生菌根担担菌纲的圆筒孢子菌丝体(约0.2克鲜重)在注射器中通过镍铬合金丝(见图4)。
    2. 用PTFE微管连接器(图3),并通过孔将一个PTFE微管插入每个注射器,并将连接器连接到气泵。调整泵的流量和PTFE微管的位置,以便在所有注射器中获得等效通气。在每个注射器的顶部添加玻璃漏斗以最小化介质的蒸发。在实验室灯下连续孵育植物和真菌长达48小时。
    3. 为了研究植物对真菌的外溢作用,在培养6和24小时48小时后取1毫升相互作用培养基,并按照我们以前的方案(Becquer)详细描述测量放射性等,,2017)。在取样之前,不要忘记在注射器中记录相互作用介质的实际体积。孵育结束时,记录每个菌丝体的鲜重,并测量其放射性(详见Becquer等人,2017)。还要每个植物;在滤纸之间进行印迹,从枝条分离出根,记录其鲜重。然后将整个植物(根+芽)置于20ml闪烁瓶中,并按照Becquer等人的描述测量放射性。 (2017)。

  7. 用 32 P标记的菌丝体计算
    1. 参考我们以前的协议,以更正原始数据(通常以每分钟cpm =计数计算),用于衰减(Becquer 等,,2017)。
    2. 计算在6,24和48小时(或任何其他潜伏期)之后使用Excel中写下列公式释放真菌的放射性净量:
      cpm t0-t6 = cpm t6 x [(60 + V bs t6 )/ 2] /> cpmt 24-t6 = cpm t24 x [(V sub)> sub> IM t24 / 2]
      cpm t24-t48 = cpm t48 x [(V sub) sub> IM t48 )/ 2]
      与:
      cpm ti =在时间i 1毫升IM中测量的每分钟计数
      在时间i采样后的交互介质的体积(ml)
      在时间i采样之前的交互介质的体积(ml)
      使用这些数据来计算真菌在不同潜伏期内损失的放射性量:
      0-6 h = cpm 0-24 h = cpm t0-t6 + cpm 0-48h = cpm t0-t6 + cpm
    3. 为了计算真菌在48小时内损失的总放射性量,将在相互作用介质(0-48小时)中测量的cpm值与在植物中测量的值相加。
    4. 将这些以前的值与在植物添加之前在真菌中测量的值相加,以获得由真菌摄取的总放射性量。
    5. 在Becquerel中使用Becquer等人给出的公式转换cpm的值。 (2017)。

数据分析

当真菌用 32 P(参见Becquer等人,2017年)标记时,每次处理的重复数应至少为6,实验应重复两次。结果可以表示为真菌损失的初始放射性百分比或每g真菌鲜重。使用Kolmogorov Smirnov测试来测试数据的正常性,并且在必要时,数据在分析之前进行平方根或log 10变换以满足方差分析的假设。孵育时间(例如,6,24或48小时)的影响以及生物条件(单株真菌与真菌的植物相比)可以使用一个方式或双向方差分析。

笔记

  1. 在海松松种子的消毒步骤中,该人必须戴上手套,以避免皮肤接触纯粹的浓缩H 2 O 2 O 2的任何燃烧。
  2. 种子必须与H 2 O 2 O 2有良好的接触才能有效消毒。
  3. 在转移到试管中时,重要的是它们的覆盖在子叶上。没有这样的幌子,幼苗会很快干涸死亡。
  4. 如果护套已经丢失,可以用过滤纸覆盖,用去离子水充分润湿,防止幼苗受到空气干燥。
  5. 根据实验所需的植物数量,可以将2个发芽种子并排放置在管中。在这种情况下,需要非常频繁地监测营养液的含量。
  6. 由于滤纸与细菌或腐生真菌污染的风险从不为零,所以通常会比所需的植物多10%。

食谱

  1. 琼脂板发芽
    1. 通过将2g D-葡萄糖溶解在1L去离子水中来制备葡萄糖溶液
    2. 取两个1升玻璃瓶,加入7.5克琼脂,每瓶500毫升葡萄糖溶液
    3. 在121℃灭菌琼脂培养基20分钟
    4. 将冷却的介质(55-60°C)倒入90 mm直径的培养皿中
  2. 0.1M Ca(OH)2 溶液
    1. 将100ml去离子水倒入250ml玻璃瓶中
    2. 加入0.74g的Ca(OH)2,并摇动
    3. 粉末可能不会完全溶解,必须用手摇动溶液以在使用前重新悬浮粉末
    4. 在室温下存放
  3. 微量元素(1000毫升)
    3.08g MnSO 4•H 2 O
    4.41g ZnSO 4•7H 2 O
    2.82g H 3 BO 3
    0.98g CuSO 4•5H 2 O
    0.29g Na 2 MoO 4•2H 2 O
    将去离子H 2 O 2加入到1000ml中,在4℃下储存
  4. 矿物盐溶液(100ml)


    所有这些解决方案都存储在4°C
  5. N1 + P溶液(1000ml)
    1. 加入1L烧杯中约500ml去离子水和以下体积的矿物溶液:
      0.2ml Ca(NO 3 3)2
      0.6ml KNO 3
      0.2 ml KCl
      0.2ml KH 2 PO 4
      0.5毫升柠檬酸铁铵/ /
    2. 添加痕量元素0.2 ml
    3. 将体积达到1升,并将pH调节至5.5
    4. 在121℃灭菌溶液20分钟
  6. CaSO 4溶液(0.2mM)
    1. 加入1L烧杯中的1000ml去离子水和34.4mg硫酸钙
    2. 仔细摇匀溶液,如果需要,将pH调至5.5,使用0.1 NH 2 SO 4或0.1 M Ca(OH)2 >
    3. 在121℃灭菌溶液20分钟
  7. 相互作用介质(3000 ml)
    1. 加入3LL烧杯中约1.5L去离子水和以下体积的矿物溶液:
      0.6ml MgSO 4
      1.5ml CaCl 2
      3.2g MES(终浓度为5mM)
      1.82克TRIS(终浓度为5 mM)
    2. 用去离子水完成3升,并用1NH 2 SO 4将pH调节至5.5。
    3. 将1.5升培养基倒入2升玻璃瓶中,并通过高压灭菌(20分钟,121℃)消毒

致谢

这项研究得到INRA(法国)的支持,通过为研究人员提供的年度资金和通过授予Adeline Becquer的年轻科学家合同(CJS)和CONACYT(墨西哥)通过授予玛格丽塔•托雷斯 - 阿基诺。该协议改编自我们之前的工作(Plassard等人,1994; Torres-Aquino等人,2017)。我们还感谢三位匿名评审员对改进方案的有益评论。

参考

  1. Becquer,A.,Torres-Aquino,M.,LeGuernevé,C.,Amenc,L.K.,Trives-segura,C.,Staunton,S.,Quiquampoix,H.and Plassard,C。(2017)。 用于研究植物 - 真菌相互作用的 的放射性标记方法。 > Bio Protoc 7(20):e2576。
  2. 凯恩,J. W. G.(2011)。 外生菌根真菌:森林土壤中磷的根系共生途径。 a>植物土壤 344:51-71。
  3. Plassard,C.,Barry,D.,Eltrop,L.和Mousain,D。(1994)。 海上松木中的硝酸盐摄入( Pinus pinaster )和外生菌根真菌(Hebeloma cylindrosporum):外生菌根共生的作用。可以J Bot 72:189-197。
  4. Plassard,C.和戴尔,B。(2010)。 菌根营养的磷营养。树生理学30 / 9):1129-1139。
  5. Smith,S.E。和Read,D.J。(2008)。 菌根共生第3版。 学术出版社。
  6. Smith,S.E.,Anderson,I.C。和Smith,F.A。(2015)。 菌根协会和磷获取:从细胞到生态系统。 年度植物评论 48:409-440。
  7. Torres-Aquino,M.,Becquer,A.,Le Guerneve,C.,Louche,J.,Amenc,L.K.,Staunton,S.,Quiquampoix,H.and Plassard,C.(2017)。 Pinus pinaster的主机植物对磷酸盐外排和多磷酸盐代谢产生特殊影响 外生菌根真菌Hebeloma cylindrosporum :放射性示踪剂,细胞学染色和 31核磁共振光谱研究植物细胞环境40(2 ):190-202。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Becquer, A., Torres-Aquino, M., Le Guernevé, C., Amenc, L. K., Trives-Segura, C., Staunton, S., Quiquampoix, H. and Plassard, C. (2017). Establishing a Symbiotic Interface between Cultured Ectomycorrhizal Fungi and Plants to Follow Fungal Phosphate Metabolism. Bio-protocol 7(20): e2577. DOI: 10.21769/BioProtoc.2577.
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