Materials and Reagents

1.5 ml Pierce microcentrifuge tubes (Thermo Scientific, catalog number: 69715)
Petri dishes (Fisher brand, Fisher Scientific, catalog number: FB0875712)
Microscope slides (Pearl, catalog number: 7101)
Microscope slide coverslips (Pearl)
1 ml Sterilin plastic transfer pipettes (Thermo Scientific, catalog number: 201C)
Surgical blade No.11 for metal scalpel 10621 (Feather, catalog number: 2976)
Household aluminum foil
Root trainers (Flight Plastic Ltd.)
Grass seeds (used here: Lolium perenne cv Samson seeds, endophyte-free; Agricom, New Zealand)
An Epichloë strain, E. festucae Fl1 (ATCC, catalog number: MYA-3407)
Autoclaved distilled water
Technical grade agar (Difco Laboratories, catalog number: DF0812-17-9)
Potato dextrose broth (Difco Laboratories, catalog number: DF0549-17-9)
L-lactate hydrate (C₃H₆O₃) (Sigma-Aldrich, catalog number: L1750)
Glycerol (HOCH₂CH(OH)CH₂OH) (Sigma-Aldrich, catalog number: G9012)
Phenol (C₆H₅OH) (Thermo Scientific, catalog number: 17914)
Trypan Blue (C₃₄H₂₄N₆O₁₄S₄Na₄) (Sigma-Aldrich, catalog number: T6146)
Chloral hydrate (Cl₃CCH(OH)₂) (Sigma-Aldrich, catalog number: C8383)
Aniline Blue W.S. (C₃₂H₂₅N₃Na₂O₉S₃) (Sigma-Aldrich, catalog number: 28631-66-5)
Tripotassium orthophosphate (K₃PO₄) (VWR, catalog number: 700001)
Safranin O (C.I. 50240) (C₂0H₁₉ClN₄) (Sigma-Aldrich, catalog number: 1159480025)
3,3’-Diaminobenzidine (DAB) (Sigma-Aldrich, catalog number: D12384)
Hydrochloric acid (HCL) (Sigma-Aldrich, catalog number: D12384)
95% Ethanol (CH₃CH₂OH) (Sigma-Aldrich, catalog number: 11727)
Sucrose (Sigma-Aldrich, catalog number: S8501)
Boric acid (BH₃O₃) (Thermo Scientific, catalog number: AC315185000)
2.4% Potato dextrose agar (see Recipes)
3% Water agar (see Recipes)
Lactophenol-Trypan Blue solution (see Recipes)
Chloral hydrate solution (see Recipes)
Aniline Blue solution (see Recipes)
Pollen germination slides (see Recipes)
Safranin solution (see Recipes)
DAB solution (see Recipes)

Equipment

100 ml glass beaker
Leica DMR microscope (camera: Leica DC500)
Autoclave
Fridge
Laminar flow cabinet
Incubator (Thermo Scientific, 3110 CO₂ Water-Jacketed Incubator)
Stereomicroscope (Leica, model: Leica M3Z)
Precellys 24 tissue disruptor (Bertin Technologies)
Stainless-steel forceps (Sigma-Aldrich, catalog number: Z168777)
Metal scalpel (Sigma-Aldrich, catalog number: S2646)

Procedure

Seedling preparation and inoculation (adapted from Latch and Christensen, 1985):
1. Seed surface sterilization:
  1. Incubate endophyte-free seeds in 50% sulfuric acid (H₂SO₄) for 30 min.
  2. Pour off the acid and soak the seeds 3 times in sterile water for 3 min each time.
  3. Incubate the seeds in 50% commercial bleach for 20 min.
  4. Pour off and soak the seeds 3 times in sterile water for 2 min each time.
  5. Dry the seeds on sterile filter paper in a laminar flow hood.
2. Seed culture:
  1. Using sterile forceps, transfer the sterile dried seeds to 4% water agar (10 seeds per plate).
  2. Incubate the plates in the dark at 22°C for 7 days.
3. Mycelium preparation for inoculation:
  1. Subculture the Epichloë strain onto a PDA plate.
  2. Incubate the plate at 20°C for 7-10 days.
4. Seedling inoculation:
  1. After 7-8 days, the seeds will germinate with etiolated shoots.
  2. Using a dissecting microscope in a laminar flow hood, make a small incision in the meristem (usually appears as a faintly visible line between the mesocotyl and the coleoptile) with a scalpel.
  3. Cut a small piece of fungal mycelium from the PDA plate (around 2 mm × 2 mm) and insert into the incision in the meristem.
  4. Incubate the seedlings in their original water agar plates at 22°C in the dark, with the plates standing on their end and the seedlings upright.
  5. Subsequent steps are described in detail (including videos and figures) in Becker et al. (2018) .
In all plant response tests, there are 4 inoculation treatments including seedlings with an incision inoculated with wild type E. festucae (“Wild Type”), mutant ΔvelA E. festucae (“ΔvelA”), a block of agar without fungi (“E- (cut)”), and seedlings without an incision but a piece of agar placed over the meristem (“E- (Uncut)”).
Staining for defense responses
Inoculated seedlings were incubated for the different plant response tests as follows:
1. Callose deposition (adapted from Knox, 1979):
  1. Impatiens walleriana pollen tubes can be used as a positive control to test whether the Aniline Blue solution is working properly.
    1. Sprinkle pollen over a freshly prepared pollen germination slide.
    2. Store the slides in a moist Petri dish (containing wet tissue paper) for at least 5 h until germination occurs.
    3. Cover the slide with a few drops of Aniline Blue solution and incubate at room temperature for 30 min.
    4. Rinse the slides twice with distilled water.
    5. Observe callose deposition in the pollen tubes using a fluorescence microscope (excitation 450-490 nm, emission >515 nm).
    6. Callose deposition appears as a yellow-green color (Figure 1A).
  2. Studying callose deposition at different time points determined that 4 days post-inoculation (DPI) was optimal for Epichloë-ryegrass associations.
    1. At 4 DPI, place the seedlings in Aniline Blue solution for 30 min at room temperature.
    2. Rinse the seedlings twice with distilled water.
    3. Remove the inoculum (fungal block, or agar block in the negative controls) from the meristem and cut out the meristem section of the seedlings including the 1.5 cm above and below.
    4. Observe callose deposition under a fluorescence microscope (excitation 450-490 nm, emission >515 nm).
    5. Callose deposition appears as a yellow-green color (Figure 1B).
  Figure 1. Callose detection. A. Using Impatiens walleriana pollen tubes as a positive control for callose detection. Left panel; pollen tubes in Aniline Blue solution, right panel; pollen tubes in only buffer without Aniline Blue. B. Callose deposition in the meristematic region of 7-d-old seedlings inoculated with wild type and ΔvelA mutant strains of E. festucae and endophyte-free cut and uncut seedlings inoculated with agar block as a control; 4 DPI stained with Aniline Blue solution. Callose deposition appears yellow-green.
2. Lignin production (adapted from Tanaka et al., 2014):
  Studying lignin production at different time points determined that 2.5 DPI was optimal for Epichloë-ryegrass associations.
  1. At 2.5 DPI, place the seedlings in Safranin solution for 10 min in the dark at room temperature.
  2. Rinse the seedlings twice with distilled water
  3. Remove the inoculum (fungal block, or agar block in the negative controls) from the meristem and cut out the meristem section of the seedlings including the 1.5 cm above and below.
  4. Observe lignin under a fluorescence microscope in brightfield.
  5. Lignin deposition appears as a red color (Figure 2).
  Figure 2. Lignin detection in the meristematic region of 7-d-old seedlings inoculated with wild type and ΔvelA mutant strains of E. festucae and endophyte-free cut and uncut seedlings inoculated with agar block as a control; 4 DPI stained with 1% Safranin O solution. Lignin deposition appears red.
3. Plant cell death (adapted from Koch and Slusarenko, 1990):
  1. The highest levels of plant cell death were observed at 7 days post-inoculation (DPI), but a time-course study can be performed from 1-12 DPI.
  2. At 7 DPI, place whole seedlings in 20 ml boiling lactophenol-Trypan Blue for 1 min. Since this solution contains corrosive phenol and requires boiling, this step should be carried out under the laminar flow cabinet. First, warm the solution in a 100-ml glass beaker over a Bunsen burner until boiling, then add the seedlings.
  3. Decolorize the stained seedlings by placing in 20 ml chloral hydrate solution for 30 min. Since chloral hydrate is toxic, this step should be carried out under the laminar flow cabinet.
  4. Rinse the seedlings twice with distilled water.
  5. Remove the inoculum (fungal block or agar block in the negative controls) from the meristem and cut out the meristem section of the seedlings including the 1.5 cm above and below.
  6. Mount the seedling sections on slides with coverslips in water.
  7. Observe the cell death response under a Nikon Ti-E inverted microscope (camera: Nikon DsRi1) in brightfield.
  8. Dead cells appear dark blue (Figure 3).
  Figure 3. Cell death detection in the meristematic region of 7-d-old seedlings inoculated with wild type and ΔvelA mutant strains of E. festucae and endophyte-free cut and uncut seedlings inoculated with agar block as a control; 4 DPI stained with lactophenol-Trypan Blue solution. Dead cells appear dark blue.
4. Hydrogen peroxide (H₂O₂) production (adapted from Thordal-Christensen et al., 1997):
  1. Detecting H₂O₂ production after the normal inoculation procedure
    1. Most H₂O₂production should be detected immediately after inoculation.
    2. Immediately after inoculation, place whole seedlings in freshly prepared DAB solution for 4 h in the dark at room temperature.
    3. Rinse the seedlings twice with distilled water
    4. Remove the inoculum (fungal block, or agar block in the negative controls) from the meristem and cut out the meristem section of the seedlings including the 1.5 cm above and below.
    5. Observe H₂O₂production under a Leica DMR microscope in brightfield.
    6. H₂O₂appears as a brown color (Figure 4A).
  2. Detecting H₂O₂ production without incision
    Since cutting the meristem produces a high level of H₂O₂, making the differentiation between treatments difficult, we optimized the methodology to measure H₂O₂in the absence of an incision.
    1. Place 1-2 mm² fungal culture on agar blocks 1 cm above the meristem.
    2. Immediately after inoculation, place whole seedlings in freshly prepared DAB solution for 4 h in the dark at room temperature.
    3. Remove the inoculum (fungal block, or agar block in the negative controls) from the meristem and cut out the meristem section of the seedlings including the 1.5 cm above and below.
    4. Observe H₂O₂production under a Leica DMR microscope in brightfield.
    5. H₂O₂appears as a brown color (Figure 4B).
Figure 4. Hydrogen peroxide (H₂O₂) detection. A. H₂O₂ production in the meristematic region of 7-d-old seedlings inoculated with wild type and ΔvelA mutant strains of E. festucae and endophyte-free cut and uncut seedlings inoculated with agar block as a control; immediately stained with DAB solution. B. H₂O₂ response 1 cm above the meristem of 7-d-old seedlings was detected immediately after inoculating seedlings using method 4b (Detecting H₂O₂ production without incision). Arbitrary scoring system for measuring the severity of the response. H₂O₂ appears brown.

Data analysis

For all detection methods and for each treatment, a range of responses can be observed because ryegrass is an out-crossing species; therefore, each plant has a different genotype. As such, we used an arbitrary categorization method based on the percentage of seedlings that show the response. For all the methods (except 4b; Detecting H₂O₂ production without incision), data analyses are based on the percentage of seedlings showing the test response (similar to Figure 1B, first on the left). As an example, the results of callose deposition in seedlings inoculated with different strains of E. festucae are presented in Figure 5.

Figure 5. Percentage of 7-d-old seedlings showing callose deposition at 4 days post-inoculation. Seedlings were inoculated with wild type and ΔvelA mutant strains of E. festucae, and endophyte-free cut and uncut seedlings were inoculated with agar block as a control. In each inoculation, 30 seedlings were inoculated. Bars represent the standard error of the mean calculated from 3 independent inoculation experiments.

To detect H₂O₂ production without incision (method 4b), a range of 5 different responses (0 to 4) was used (Figure 4B) due to the sensitivity of the method. The percentage of seedlings showing an H₂O₂ response can be visualized in Figure 6.

Figure 6. Percentage of 7-d-old seedlings showing the production of hydrogen peroxide (H₂O₂). The H₂O₂ response 1-cm above the meristem of 7-d-old seedlings was detected immediately after inoculating seedlings using method 4b (detecting H₂O₂ production without incision) with wild type (WT) and ΔvelA mutant strains of E. festucae and agar block as a control (E-). In each inoculation, 30 seedlings were inoculated. Numbers 1-4 in the legend represent arbitrary categorization of the H₂O₂ responses represented in Figure 4B.

Notes

Three independent inoculation tests were performed for each method.
The number of seedlings used for each test should be greater than 30, and increasing the number of seedlings can help to reduce the variability of the responses that can result from differences in genetic background of the seeds.
In all the detection methods, we normally stain whole seedlings and cut out the meristem region (region of interest) for microscopy. This reduces host responses due to wounding that can interfere with microscopy.

Recipes

2.4% potato dextrose agar
1. Dissolve 24 g potato dextrose broth and 15 g agar in 1 L distilled water
2. Autoclave for 30 min at 121°C and pour 25 ml solution per Petri dish
3. Plates can be stored in the fridge for up to one month
3% water agar
1. Dissolve 30 g agar in 1 L distilled water
2. Autoclave for 30 min at 121°C and pour 25 ml solution per Petri dish
3. Plates can be stored in the fridge for up to one month
Lactophenol-trypan blue solution
1. Dissolve 10 mg trypan blue in 10 ml distilled water and add 10 ml lactic acid (98%), 10 ml glycerol, and 10 ml phenol
2. The solution can be stored in the dark in the fridge for up to 3 months
Chloral hydrate solution
1. Dissolve 2.5 g chloral hydrate in 1 ml distilled water
2. The solution can be stored in the fridge for up to 1 month
Aniline blue solution
1. Dissolve 0.1 g aniline blue and 2.3 g tripotassium orthophosphate in 100 ml distilled water
2. Place the solution in the dark for 24 h until it becomes colorless
3. The solution can be stored in the dark in the fridge for up to 3 months
Pollen germination slides
1. Dissolve 10 g sucrose, 1 g agar, and 8 mg boric acid in 100 ml distilled water
2. Autoclave for 30 min at 121°C and drop on a slide to make a flat film
3. Slides can be stored in the fridge for up to one week
Safranin solution
1. Dissolve 1 g Safranin O (C.I. 50240) in 100 ml distilled water
2. The solution can be stored in the dark in the fridge for up to 3 months
DAB solution
1. Dissolve 10 mg 3,3’-diaminobenzidine (DAB) in 10 ml distilled water
2. Adjust the pH to 3.8 using HCl
3. The solution should be prepared fresh for each test

Acknowledgments

This research was funded by grants from the Royal Society of New Zealand Marsden Fund, contract AGR1002, and the New Zealand Strategic Science Investment Fund, contract A20067. We thank Adrian Turner (Microscopy & Graphics Unit, University of Auckland), C.R. Voisey, W.R. Simpson, W. Mace, and A. deBonth for technical assistance (Forage Improvement, AgResearch Grasslands), and Biotelliga Ltd. for providing laboratory space.

Competing interests

The authors declare no conflicts of interest.

References

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