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Jun 2018
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Histological Methods to Detect Early-stage Plant Defense Responses during Artificial Inoculation of Lolium perenne with Epichloë festucae
羊茅人工接种多年生黑麦草过程中植物早期防御反应的组织学检测   

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Abstract

Epichloë species form agriculturally important symbioses with many cool season grasses. To study these symbioses, such as the interaction of Epichloë festucae with perennial ryegrass (Lolium perenne), host plants can be infected by artificial inoculation of etiolated seedlings. This inoculation is performed by placing mycelium into an incision in the meristem, as previously described by Latch and Christensen (1985). In recent years, this method has been broadly used to study this interaction at the molecular level using different Epichloë festucae mutants that can cause incompatible interactions. We have developed and adapted methods to study four of the most important host plant responses to infection, including cell death, callose deposition, lignin production, and hydrogen peroxide (H2O2) production, which are useful in defining the host response to infection at a very early time point.

Keywords: Endophytic fungi (内生真菌), Plant defense (植物防御), Mutualism (共生), Symbiotic (共生的), Plant-fungal interactions (植物-真菌相互作用), ROS production (活性氧自由基生成)

Background

Although artificial inoculation of perennial ryegrass (Lolium perenne) is broadly used to study its interaction with Epichloë festucae, there exists no comprehensive protocol for the evaluation of responses to fungal infection at an early post-inoculation time point, which likely defines the future of this interaction. In our recent work (Rahnama et al., 2018), we adapted methods to study four of the most important host plant responses during plant-fungal interactions including cell death, callose deposition, lignin production, and hydrogen peroxide (H2O2) production.


In response to invading microbes, one of the early plant actions is the production of different types of reactive oxygen species (ROS), including H2O2, which act as an antimicrobial agent to protect the plant against invading microbes and are one of the first signals to induce other plant responses (Walters, 2003). In addition, by depositing callose and lignin, plants remodel their cell wall at the infection site to stop invading fungi (Luna Diez et al., 2010; Zipfel, 2009). As a late defense response, plant cells surrounding the infection site undergo programmed cell death (hypersensitive response) to limit available food for the invading microbe (Lo Presti et al., 2015). To initiate and maintain a successful symbiotic lifestyle, Epichloë endophytes must somehow suppress or avoid these different host defense responses; although, very little is known about how this occurs. Developing methods to detect these responses will aid in the understanding of this system, especially when applied to the study of mutant strains that compromise symbiosis.


To stain for callose deposition, we adapted an Aniline Blue staining method (Knox, 1979) that was originally used for the detection of callose in pollen tubes but has also been used in different fungal-plant interactions such as powdery mildew infection of Arabidopsis (Ellinger et al., 2013). Besides aniline blue, Toluidine Blue O has also been used in other systems such as guava root infection with Fusarium (Gupta et al., 2012). In addition to staining methods, there exist non-staining methods using immunofluorescence that detect callose at the micro-level in internal cell sites such as plasmodesmata (Pendle and Benitez-Alfonso, 2015).


To detect lignin deposition, we used a Safranin solution adapted from a study on maize infection by Ustilago maydis (Tanaka et al., 2014); however, Safranin is broadly used in other systems such as Fusarium infection of different cereals (Knight et al., 2011). Moreover, the Wiesner (phloroglucinol-HCl) reaction (Pomar et al., 2002) is another method to study lignin deposition in plant-fungal interactions.


To visualize cell death responses, Trypan Blue staining is the most widely used method, for which we adapted the protocol used to study Arabidopsis infection with downy mildew (Koch and Slusarenko, 1990). Recently, a non-toxic method has also been suggested, which uses red light imaging (Landeo Villanueva et al., 2021).


There are several methods to detect H2O2, one of the basic methods for which is the detection of the oxidization of small molecules, which we also used here. This method is based on visualizing the color change of 3,3’-diaminobenzidine (DAB) after being oxidized by H2O2. We adapted this method from a study on barley infection with powdery mildew (Thordal-Christensen et al., 1997). Other recent methods to study H2O2 have used genetically encoded green fluorescent protein (GFP)-based probes that react directly with H2O2 (reviewed in Winterbourn, 2018).


Here, we demonstrate that these methods are useful for defining the response of perennial ryegrass to infection with Epichloë endophyte at a very early time point. Using these methods in time-point studies, we defined the most suitable time to measure these responses post-inoculation. As such, these methods are useful for studying compatible and incompatible interactions of symbiotic fungi with their host plants during the early stages of infection (Rahnama et al., 2018 and 2019). Although these methods are routinely used for other fungal-plant interactions, our adaption of them to study the early inoculation stages of Epichloë-grass interactions is novel. These methods can be used in other grass-endophyte interactions that use a similar inoculation technique.

Materials and Reagents

  1. 1.5 ml Pierce microcentrifuge tubes (Thermo Scientific, catalog number: 69715)

  2. Petri dishes (Fisher brand, Fisher Scientific, catalog number: FB0875712)

  3. Microscope slides (Pearl, catalog number: 7101)

  4. Microscope slide coverslips (Pearl)

  5. 1 ml Sterilin plastic transfer pipettes (Thermo Scientific, catalog number: 201C)

  6. Surgical blade No.11 for metal scalpel 10621 (Feather, catalog number: 2976)

  7. Household aluminum foil

  8. Root trainers (Flight Plastic Ltd.)

  9. Grass seeds (used here: Lolium perenne cv Samson seeds, endophyte-free; Agricom, New Zealand)

  10. An Epichloë strain, E. festucae Fl1 (ATCC, catalog number: MYA-3407)

  11. Autoclaved distilled water

  12. Technical grade agar (Difco Laboratories, catalog number: DF0812-17-9)

  13. Potato dextrose broth (Difco Laboratories, catalog number: DF0549-17-9)

  14. L-lactate hydrate (C3H6O3) (Sigma-Aldrich, catalog number: L1750)

  15. Glycerol (HOCH2CH(OH)CH2OH) (Sigma-Aldrich, catalog number: G9012)

  16. Phenol (C6H5OH) (Thermo Scientific, catalog number: 17914)

  17. Trypan Blue (C34H24N6O14S4Na4) (Sigma-Aldrich, catalog number: T6146)

  18. Chloral hydrate (Cl3CCH(OH)2) (Sigma-Aldrich, catalog number: C8383)

  19. Aniline Blue W.S. (C32H25N3Na2O9S3) (Sigma-Aldrich, catalog number: 28631-66-5)

  20. Tripotassium orthophosphate (K3PO4) (VWR, catalog number: 700001)

  21. Safranin O (C.I. 50240) (C20H19ClN4) (Sigma-Aldrich, catalog number: 1159480025)

  22. 3,3’-Diaminobenzidine (DAB) (Sigma-Aldrich, catalog number: D12384)

  23. Hydrochloric acid (HCL) (Sigma-Aldrich, catalog number: D12384)

  24. 95% Ethanol (CH3CH2OH) (Sigma-Aldrich, catalog number: 11727)

  25. Sucrose (Sigma-Aldrich, catalog number: S8501)

  26. Boric acid (BH3O3) (Thermo Scientific, catalog number: AC315185000)

  27. 2.4% Potato dextrose agar (see Recipes)

  28. 3% Water agar (see Recipes)

  29. Lactophenol-Trypan Blue solution (see Recipes)

  30. Chloral hydrate solution (see Recipes)

  31. Aniline Blue solution (see Recipes)

  32. Pollen germination slides (see Recipes)

  33. Safranin solution (see Recipes)

  34. DAB solution (see Recipes)

Equipment

  1. 100 ml glass beaker

  2. Leica DMR microscope (camera: Leica DC500)

  3. Autoclave

  4. Fridge

  5. Laminar flow cabinet

  6. Incubator (Thermo Scientific, 3110 CO2 Water-Jacketed Incubator)

  7. Stereomicroscope (Leica, model: Leica M3Z)

  8. Precellys 24 tissue disruptor (Bertin Technologies)

  9. Stainless-steel forceps (Sigma-Aldrich, catalog number: Z168777)

  10. Metal scalpel (Sigma-Aldrich, catalog number: S2646)

Procedure

  1. 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)”).


  2. 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 H2O2 production 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 H2O2 production under a Leica DMR microscope in brightfield.

        6. H2O2 appears 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 H2O2 in 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 H2O2 production under a Leica DMR microscope in brightfield.

        5. H2O2 appears 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

  1. Three independent inoculation tests were performed for each method.

  2. 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.

  3. 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

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. 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

  1. Latch, G. C. M. and Christensen, M. J. (1985). Artificial infection of grasses with endophytes. Ann Appl Biol 107: 17-24.
  2. Becker, Y., Green, K. A., Scott, B. and Becker, M. (2018). Artificial inoculation of Epichloë festucae into Lolium perenne, and visualisation of endophytic and epiphyllous fungal growth. Bio-protocol 8(17): e2990.
  3. Ellinger, D., Naumann, M., Falter, C., Zwikowics, C., Jamrow, T., Manisseri, C., Somerville, S. C. and Voigt, C. A. (2013). Elevated early callose deposition results in complete penetration resistance to powdery mildew in Arabidopsis.Plant Physiol 161(3): 1433-1444.
  4. Gupta, V. K., Misra, A. K. and Pandey, B. K. (2012). Histological changes in guava root during wilting induced by Fusarium spp. Arch Phytopathology Plant Protect 45(5): 570-573.
  5. Knox, R. B. (1979). Pollen and allergy. Studies in Biology. No. 107. Arnold, London.
  6. Koch, E. and Slusarenko, A. (1990). Arabidopsis is susceptible to infection by a downy mildew fungus. Plant Cell 2(5): 437-445.
  7. Lo Presti, L., Lanver, D., Schweizer, G., Tanaka, S., Liang, L., Tollot, M., Zuccaro, A., Reissmann, S. and Kahmann, R. (2015). Fungal effectors and plant susceptibility. Annu Rev Plant Biol 66: 513-545.
  8. Luna, E., Pastor, V., Robert, J., Flors, V., Mauch-Mani, B. and Ton, J. (2011). Callose deposition: a multifaceted plant defense response. Mol Plant Microbe Interact 24(2): 183-193.
  9. Pendle, A. and Benitez-Alfonso, Y. (2015). Immunofluorescence detection of callose deposition around plasmodesmata sites. In: Heinlein, M. (Ed.). Plasmodesmata. Methods in Molecular Biology (Methods and Protocols), vol 1217, pp95-104.Humana Press, New York, NY.
  10. Pomar, F., Merino, F. and Barcelo, A. R. (2002). O-4-Linked coniferyl and sinapyl aldehydes in lignifying cell walls are the main targets of the Wiesner (phloroglucinol-HCl) reaction. Protoplasma 220(1-2): 17-28.
  11. Rahnama, M., Johnson, R. D., Voisey, C. R., Simpson, W. R. and Fleetwood, D. J. (2018). The global regulatory protein vela is required for symbiosis between the endophytic fungus Epichloë festucae and Lolium perenne. Mol Plant Microbe Interact 31(6): 591-604.
  12. Rahnama, M., Maclean, P., Fleetwood, D. J. and Johnson, R. D. (2019). The LaeA orthologue in Epichloë festucae is required for symbiotic interaction with Lolium perenne. Fungal Genet Biol 129: 74-85.
  13. Tanaka, S., Brefort, T., Neidig, N., Djamei, A., Kahnt, J., Vermerris, W., Koenig, S., Feussner, K., Feussner, I. and Kahmann, R. (2014). A secreted Ustilago maydis effector promotes virulence by targeting anthocyanin biosynthesis in maize. Elife 3: e01355.
  14. Thordal-Christensen, H., Zhang, Z., Wei, Y. and Collinge, D. B. (1997). Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley—powdery mildew interaction. The Plant J 11(6): 1187-1194.
  15. Landeo Villanueva, S., Malvestiti, M. C., van Ieperen, W., Joosten, M. and van Kan, J. A. L. (2021). Red light imaging for programmed cell death visualization and quantification in plant-pathogen interactions. Mol Plant Pathol 22(3): 361-372.
  16. Walters, D. R. (2003). Polyamines and plant disease. Phytochemistry 64(1): 97-107.
  17. Winterbourn, C. C. (2018). Biological Production, Detection, and Fate of Hydrogen Peroxide. Antioxid Redox Signal 29(6): 541-551.
  18. Zipfel, C. (2009). Early molecular events in PAMP-triggered immunity. Curr Opin Plant Biol 12(4): 414-420.

简介

[摘要] Epichloë物种与许多凉季草形成农业上重要的共生体。为了研究这些共生,如互动的Epichlo ë festucae与多年生黑麦草(黑麦草),寄主植物可通过黄化幼苗人工接种感染。此接种是通过将菌丝体成分生组织中的切口执行,如previo usly描述了ð由锁存和Christensen(1985)。近年来,这种方法已经被广泛地用于研究在使用不同的分子水平上这种互动Epichlo ë festucae突变,可导致不兼容的相互作用。我们已经开发并适于的方法来研究的最重要的宿主植物对感染,包括细胞死亡,胼胝质沉积,木质素生产的4 ,和过氧化氢(H 2 ö 2 )的生产,这是有用的在defin荷兰国际集团宿主对在很早的时间点感染。


[背景]虽然多年生黑麦草的人工接种(黑麦草)被广泛地用于研究其相互作用Epichlo ë festucae ,有存在不全面的协议为评价对真菌感染的反应在早期交-接种时间点,这可能定义互动的未来。在我们最近的工作(Rahnama 。等,2018) ,我们调整方法的植物中研究最重要的寄主植物反应四-真菌相互作用,包括细胞死亡,胼胝质沉积,木质素生产,和过氧化氢(H 2 Ø 2 )生产。

响应于入侵的微生物,早期植物的一个动作是在produc和灰不同类型的活性氧(ROS) ,含H的2 ö 2 ,其作为抗微生物剂,以保护植物对抗入侵的微生物,并且其中一个诱导其他植物反应的第一个信号(Walters,2003)。另外,通过沉积call质和木质素,植物在感染部位重塑了ir细胞壁,以停止入侵真菌(Luna Diez等,2010; Zipfel ,2009)。作为晚期防御反应,感染部位周围的植物细胞会经历程序性的细胞死亡(超敏反应),以限制入侵微生物的可用食物(Lo Presti等人,2015)。为了启动和维持成功的共生生活方式,Epichloë内生真菌必须以某种方式抑制或避免这些不同的宿主防御反应; 一个lthough ,很少有人知道怎么发生这种情况。d eveloping方法来检测这些反应将有助于在理解荷兰国际集团的这一系统,特别是当应用到了学习的突变株妥协共生。

为了对call的沉积进行染色,我们采用了A niline B lue染色方法(Knox,1979年),该方法最初用于检测花粉管中的ose ,但也已用于不同的真菌-植物相互作用中,例如白粉病的侵染。拟南芥(Ellinger et al。,2013)。除了苯胺蓝以外,T oluidine B lue O还被用于其他系统,例如番石榴根尖感染镰刀菌(Gupta et al。,2012)。除了染色方法,有存在用免疫荧光非染色方法在于在检测胼胝质的微-电平如胞间连丝(彭德尔在内部小区站点和贝尼特斯-阿方索,2015)。

为了检测沉积木质素,我们使用一个番红溶液适于从一个研究ø Ñ由玉米感染玉蜀黍黑粉菌(田中等人,2014); 然而,番红被广泛地在其它系统中使用,例如镰孢属infecti上的不同谷类(奈特等人,2011)。此外,维斯纳(间苯三酚-HCl)反应(Pomar等,2002)是研究木质素在植物-真菌相互作用中沉积的另一种方法。

为了可视化细胞死亡反应,T rypan B lue染色是使用最广泛的方法,为此我们采用了用于研究霜霉病的拟南芥感染的方案(Koch和Slusarenko,1990)。近来,一种无毒方法已也被建议,其中我们上课红光成像(Landeo维拉纽瓦等人,202 1 )。

有几种方法来检测ħ 2 ö 2 ,直径:NE的基本方法对于其是将检测的离子的小分子的氧化,由此我们也用在这里。该方法基于可视化被H 2 O 2氧化后的3,3'-二氨基联苯胺(DAB)的颜色变化。我们改编自研究O此方法Ñ大麦感染白粉病(Thordal-克里斯坦森等人,1997)。研究H 2 O 2的其他最新方法使用了基于遗传编码的绿色荧光蛋白(GFP)的探针,这些探针可直接与H 2 O 2反应(在Winterbourn进行综述,2018)。

在这里,我们证明,这些方法一重新有用的defin荷兰国际集团多年生黑麦草对感染的反应稻香内生菌在很早的时间点。在使用时这些方法-点的研究,我们确定了最适合的时间来测量这些反应后-接种。因此,这些方法可用于研究与宿主植物共生真菌的相容和不相容的相互作用有用期间感染的早期阶段(Rahnama等人,2018和2019)。尽管这些方法通常用于其他真菌-植物相互作用,但我们对研究埃奇克洛-草相互作用的早期接种阶段的适应性是新颖的。这些方法可以在其他草使用-内生菌使用交互一个类似的接种技术。

关键字:内生真菌, 植物防御, 共生, 共生的, 植物-真菌相互作用, 活性氧自由基生成

材料和试剂
1.5毫升皮尔斯米icrocentrifuge吨ubes物(Thermo Scientific,目录号:69715)
陪替氏d ishes(Fisher牌,Fisher Scientific公司,目录号:FB0875712)
显微镜载玻片(珍珠,目录号:7101)
显微镜幻灯片盖玻片(珍珠)
1毫升Sterilin p拉斯蒂克吨转让(BOT)p ipettes物(Thermo Scientific,目录号:201C)
11号手术刀,用于金属手术刀10621(羽毛,目录号:2976)
家用铝箔
根训练员(飞行塑胶有限公司。)
草种子(这里使用:黑麦草品种参孙种子,内生真菌-免费; Agricom,新西兰)
Epichloë菌株,E。festucae Fl1(ATCC,目录号:MYA-3407)
高压灭菌蒸馏水
技术克RADE一个GAR(Difco实验室,目录号:DF0812-17-9)
马铃薯d extrose b罗斯(Difco实验室,目录号:DF0549-17-9)
L-乳酸水合物(C 3 H 6 O 3 )(Sigma-Aldrich,目录号:L1750)
甘油(HOCH 2 CH(OH)CH 2 OH)(Sigma-Aldrich,目录号:G9012)
苯酚(C 6 H 5 OH)(Thermo Scientific,目录号:17914)
台盼蓝(C 34 H 24 N 6 O 14 S 4 Na 4 )(Sigma-Aldrich,目录号:T6146)
氯水合物(Cl 3 CCH(OH)2 )(Sigma-Aldrich,目录号:C8383)
苯胺蓝WS(C 32 H 25 N 3 Na 2 O 9 S 3 )(Sigma-Aldrich,目录号:28631-66-5)
三钾ø rthophosphate(K 3 PO 4 )(VWR,目录号:700001)
番红O(CI 50240)(C 2 0H 19 ClN 4 )(Sigma-Aldrich,目录号:1159480025)
3,3'- D氨基联苯胺(DAB)(Sigma-Aldrich,目录号:D12384)
盐酸(HCL)(Sigma-Aldrich,目录号:D12384)
95%乙醇(CH 3 CH 2 OH)(Sigma-Aldrich,目录号:11727)
蔗糖(Sigma-Aldrich,目录号:S8501)
硼酸(BH 3 O 3 )(Thermo Scientific,目录号:AC315185000)
2.4%P otato葡萄糖琼脂(见配方)
3%w ^亚特琼脂(见配方)
Lactophenol- Ť rypan乙略溶液(见配方)
氯水合物溶液(请参阅配方)
苯胺乙略溶液(见配方)
花粉萌发幻灯片(请参阅食谱)
番红花溶液(请参阅食谱)
DAB解决方案(请参阅食谱)


设备


100毫升玻璃烧杯
徕卡DMR显微镜(相机:徕卡DC500)
高压釜
冰箱
层流柜
保温箱(Thermo Scientific ,3110 CO 2水-夹套保温箱)
体视显微镜(Leica,型号:Leica M3Z)
Precellys 24组织破坏者(Bertin Technologies)
不锈钢-钢镊子(Sigma-Aldrich公司,目录号:Z168777)
金属手术刀(Sigma-Aldrich,目录号:S2646)


程序


苗的准备和接种(改编自Latch和Christensen,1985年):
种子表面灭菌:
将不含内生菌的种子在50%硫酸(H 2 SO 4 )中孵育30分钟。
倒掉的酸和将种子浸泡3次在无菌水中,每次3分钟。
将种子在50%商业漂白剂中孵育20分钟。
倒出种子,将种子浸入无菌水中3次,每次2分钟。
干燥的无菌滤纸上在种子层流罩。
种子培养:
使用无菌镊子,转移的无菌干燥的种子至4%水琼脂(每板10个种子)。
孵育的在黑暗中板在22℃下7天。
接种菌丝体的准备工作:
将Epichloë菌株传代到PDA平板上。
在20°C下孵育平板7-10天。
接种苗:
7-8天后,将种子将与黄化苗发芽。 
使用在层流橱中的解剖显微镜,做一个小切口在分生组织(通常会出现之间的依稀可见线的中胚轴和所述胚芽鞘)用手术刀。
从PDA板上切下一小块真菌菌丝体(约2 mm × 2 mm),并将其插入到分生组织的切口中。 
孵育的在它们的原始水琼脂平板的幼苗在黑暗中在22℃ ,用该板站在他们的端部和所述幼苗直立。
Becker等人详细描述了后续步骤(包括视频和图表)。,(2018)。
在所有植物响应测试,有4个接种处理包括具有切口的幼苗和野生型接种E. festucae (“野生型”),突变体Δ维拉E. festucae (“ Δ维拉”),琼脂机智块豪特真菌(“E-(切断)”) ,并且在没有切口苗但一块琼脂地方的d在分生组织(“E-(足本)”)。


染色德芬小号Ë响应
接种的幼苗培养的不同植物应答测试如下:


胼胝质沉积(公元一诺克斯,1979年PTED):
非洲凤仙花花粉管可以用作阳性对照,以测试是否该苯胺乙略溶液是否正常工作。
将花粉撒在刚准备好的花粉发芽玻片上。
存储的在潮湿的幻灯片P ETRI培养皿(含有直到发芽发生至少5小时的湿纸巾)。
用几滴A niline B lue溶液覆盖玻片,并在室温下孵育30分钟。
冲洗幻灯片TW冰用蒸馏水。
使用荧光显微镜观察花粉管中的ose质沉积(激发450-490 nm,发射> 515 nm)。
胼胝质沉积显示为黄色-绿色颜色(图1A)。
在不同时间点研究的胼胝质沉积中确定4天后-接种(DPI)为最佳稻香-黑麦草关联。
在4 DPI,放置在苗甲niline乙在室温下30分钟略溶液。
用蒸馏水冰洗幼苗。
除去接种物(真菌块,从分生组织和切割或在阴性对照琼脂块)从秧苗的分生组织部分包括所述1.5厘米上方和下方。
在荧光显微镜下观察call质沉积(激发450-490 nm,发射> 515 nm)。
胼胝质沉积似乎为黄色-绿色颜色(图1B)。




图1.毛刺检测。答:使用凤仙花花粉管作为call检测的阳性对照。左面板;花粉管中甲niline乙略溶液,右图; 花粉管仅在无A苯胺B缓冲液的缓冲液中。在B.胼胝质沉积所述的7-d龄幼苗分生组织区域接种用野生型和Δ维拉的突变菌株E. festucae和内生真菌-自由切割,并用琼脂块作为接种未切割秧苗一个控制; 4 DPI用A niline B lue溶液染色。ose质沉积出现黄色-绿色。           



木质素生产(改编自Tanaka等,2014):
研究在不同时间点的木质素生产确定2.5 DPI是用于最佳稻香-黑麦草关联。


在2.5 DPI,放置在幼苗番红溶液在黑暗中10分钟,在室温下。
用蒸馏水冰洗幼苗
除去接种物(真菌块,从分生组织和切割或在阴性对照琼脂块)从秧苗的分生组织部分包括1.5厘米上方和下方。
在明场的荧光显微镜下观察木质素。
木质素沉积显示为一红色彩色(图2)。




图2 。木质素检测在所述的7-d龄幼苗分生组织区域接种用野生型和Δ维拉的突变菌株E. festucae和内生真菌-自由切割,并用琼脂块作为接种未切割秧苗一个控制; 4 DPI用1%番红O溶液染色。木质素沉积物显示为红色。


植物细胞死亡(公元一从Koch和Slusarenko,1990年PTED):
为H ighest植物细胞死亡的水平,7天后观察-接种(DPI) ,但时间-可以学习课程进行1-12 DPI。
在7 DPI下,将整个幼苗放在20 ml沸腾的乳酚-T rypan B lue中煮1分钟。由于这种解决方案包含小号腐蚀性苯酚和需要沸腾,该步骤应当进行下的层流柜。首先,预热在该溶液100 -毫升玻璃烧杯中在本生灯至沸腾,然后添加苗。
脱色的通过在20毫升放置染色苗氯醛30分钟水合物溶液。由于水合氯醛是有毒的,该步骤应当进行下的层流柜。
用蒸馏水冰洗幼苗。
从分生组织中除去接种物(阴性对照中的真菌块或琼脂块),并切下幼苗的分生组织部分,包括上下1.5 cm。
将幼苗部分安装在装有盖玻片的载玻片上。
在明场的尼康Ti-E倒置显微镜(相机:Nikon DsRi1)下观察细胞死亡反应。
d EA d细胞出现深蓝色(图3)。




图3.细胞死亡检测在所述的7-d龄幼苗分生组织区域接种用野生型和Δ维拉的突变菌株E. festucae和内生真菌-自由切割,并用琼脂块作为接种未切割秧苗一个控制; 4 DPI用乳酚-T rypan B lue溶液染色。d EA d细胞出现深蓝色。


过氧化氢(H 2 ö 2 )生产(AD一个从Thordal-克里斯坦森PTED等人,1997):
ħ检测2 ö 2制造后的正常接种程序
接种后应立即检测出大多数H 2 O 2的产生。
接种后,立即将整株幼苗置于新鲜准备的DAB溶液中,在黑暗中于室温放置4小时。
用蒸馏水冰洗幼苗
除去接种物(真菌块,从分生组织和切割或在阴性对照琼脂块)从秧苗的分生组织部分包括1.5厘米上方和下方。
在明场的Leica DMR显微镜下观察H 2 O 2的产生。
H 2 O 2显示为棕色(图4A)。
无需切口即可检测H 2 O 2的产生
由于切割分生组织会产生高水平的H 2 O 2,从而使治疗之间的区分变得困难,因此我们优化了在没有切口的情况下测量H 2 O 2的方法。


地点1 - 2毫米2在琼脂块分生组织上方1cm处真菌培养。
接种后,立即将整株幼苗置于新鲜准备的DAB溶液中,在黑暗中于室温放置4小时。
除去接种物(真菌块,从分生组织和切割或在阴性对照琼脂块)从秧苗的分生组织部分包括1.5厘米上方和下方。
在明场的Leica DMR显微镜下观察H 2 O 2的产生。
ħ 2 ö 2显示为一棕色(图4B)。




图4.过氧化氢(H 2 O 2 )检测。A.ħ 2 ö 2生产中的7- d龄幼苗分生组织区域接种用野生型和Δ维拉的突变菌株E. festucae和内生真菌-自由切割,并用琼脂块作为接种未切割秧苗一个控制; 立即用DAB溶液染色。B.在使用方法4b接种幼苗后立即检测到7d龄幼苗的分生组织上方1cm处的H 2 O 2响应(检测没有切口的H 2 O 2产生)。任意评分系统,用于测量所述的严重性的响应。H 2 O 2呈现棕色。


数据分析


对于所有的检测方法和对于每个处理,可以观察到的响应的范围,因为黑麦草是一个出-交叉物种; 因此,每种植物都有不同的基因型。因此,我们根据显示出响应的幼苗百分比使用d任意分类方法。对于所有的方法(除了图4b ; d etectingħ 2 ö 2生产无切口),数据分析基于苗表示测试响应(类似的百分比到图URE 1B,第一ø n中的左侧)。作为一个例子,胼胝质沉积与不同菌株接种的幼苗结果E. festucae是在呈现˚F igure 5。


图5的7-d龄幼苗表示第4天的胼胝质沉积百分比后-接种。幼苗用野生型和接种Δ维拉的突变株E. festucae ,和内生真菌-自由切割和未切割的幼苗被用琼脂块作为接种一个控制。每次接种中,接种了30株幼苗。条形表示从3个独立的接种实验计算得出的平均值的标准误差。



到检测ħ 2 ö 2生产无切口(米ethod 4B ),的范围内的5个不同的响应(0〜4),使用(图URE 4B)由于M的灵敏度ethod。该p苗表示H的ercentage 2 ö 2响应可以在被可视˚F igure 6。




图6.百分比的7-d龄幼苗示出了生产的过氧化氢(H 2 ö 2 )。的ħ 2 ö 2响应1 -厘米以上的7- d龄幼苗分生组织被接种幼苗后立即检测到使用方法图4b (检测ħ 2 ö 2生产无切口)与野生型(WT)和Δ维拉的突变株E. festucae和琼脂块作为一个控制(E-)。每次接种中,接种了30株幼苗。数小号1-4在图例中表示的H的任意分类2个ö 2响应图表示URE 4B。


笔记


每种方法进行了三个独立的接种测试。
n个用于各试验苗棕土应大于30 ,和增加的幼苗的数目可以帮助减少可能是由遗传背景的差异的响应的可变性的种子。
在所有的检测方法,我们通常染色全苗和切出的分生组织区域(关注区域)的显微镜。由于伤口可能会干扰显微镜检查,因此减少了宿主反应。


菜谱


2.4%马铃薯葡萄糖琼脂
将24克马铃薯葡萄糖肉汤和15克琼脂溶解在1升蒸馏水中
高压釜在121 30分钟℃,倾25毫升溶液每P ETRI菜
盘子可以在冰箱中保存长达一个月
3%琼脂水
溶解30克琼脂在1升蒸馏水
高压釜在121 30分钟℃,倾25毫升溶液每P ETRI菜
盘子可以在冰箱中保存长达一个月
乳酚-锥虫蓝溶液
溶解10mg的台盼蓝在10毫升蒸馏水中并添加10米升乳酸(98%),将10毫升甘油,和10毫升酚
在S olution可以存储在黑暗中在冰箱里长达3个月
氯水合物溶液
将2.5克水合氯醛溶于1毫升蒸馏水
在S olution可以储存在冰箱里长达1个月
苯胺蓝溶液
将0.1 g苯胺蓝和2.3 g正磷酸三钾溶解在100 ml蒸馏水中
将溶液在黑暗中放置24小时,直到变成无色
在S olution可以存储在黑暗中在冰箱里长达3个月
花粉萌发幻灯片
溶解10克蔗糖,1克琼脂,并在100毫升8毫克硼酸蒸馏水
在121°C下高压灭菌30分钟,然后将其放在载玻片上以制成平膜
幻灯片可在冰箱中保存长达一周
番红花溶液
将1 g番红花O(CI 50240)溶于100 ml蒸馏水中
在S olution可以存储在黑暗中在冰箱里长达3个月
DAB解决方案
将10 mg 3,3'-二氨基联苯胺(DAB)溶于10 ml蒸馏水中
使用HC将pH调节至3.8升
每次测试均应准备新鲜的溶液


致谢


这项研究是由出资资助小号来自新西兰马斯登基金合同AGR1002皇家学会,以及新西兰战略科学投资基金,合同A20067。我们感谢阿德里安·特纳(显微镜和图形单元,奥克兰大学),CR的Voisey,WR辛普森,W.锤,和A. deBonth技术援助(牧草改良,草原AgResearch)和Biotelliga有限公司。用于提供实验室空间。


利益争夺


作者宣称没有冲突小号的兴趣。


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引用:Rahnama, M., Fleetwood, D. J. and Johnson, R. D. (2021). Histological Methods to Detect Early-stage Plant Defense Responses during Artificial Inoculation of Lolium perenne with Epichloë festucae. Bio-protocol 11(9): e4013. DOI: 10.21769/BioProtoc.4013.
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