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Jul 2019

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Maintenance and Quantitative Phenotyping of the Oomycete-plant Model Pathosystem Hyaloperonospora arabidopsidisArabidopsis
卵菌-植物模型病理系统(活体营养型卵菌–拟南芥)的维持和定量表型分析   

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Abstract

The interaction between the host plant Arabidopsis thaliana (Arabidopsis) and the oomycete Hyaloperonospora arabidopsidis (Hpa) is an established model system for the study of an obligate biotrophic downy mildew interaction. The evaluation of the developmental success of Hpa is often based on the quantification of reproductive structures that are formed on the surface of leaves, such as the sporangiophores or the conidiospores they carry. However, the structural basis of this interaction lies within the plant tissue and, in particular, the haustoria that form inside plant cells. Therefore, valuable additional information about the performance and compatibility of the downy mildew interaction can be gained by light microscopical inspection of the hyphal and haustorial shape inside the plant tissue and within plant cells respectively. Here we describe a protocol for the visualization and quantification of morphological phenotypes inside the plant. While we focus specifically on the quantification of haustorial shape variants, the protocol can easily be adapted for the quantification of other morphological features such as hyphal deformations, or oogonia frequency. By including and refining already existing protocols from a variety of sources, we assembled the entire experimental pipeline for the Arabidopsis Hpa bioassay to provide a practical guide for the initial setup of this system in the laboratory. This pipeline includes the following steps: A) growing Arabidopsis, B) Hpa propagation and strain maintainance C) Hpa inoculation and incubation D) staining of plant tissues for visualization of the pathogen and E) an introduction of the Keyence VHX microscope and Fiji plugin of ImageJ for the quantification of structures of interest. While described here for Arabidopsis and Hpa, the protocol steps B-E should be easily adjustable for the study of other plant-oomycete pathosystems.

Keywords: Arabidopsis (拟南芥), Hyaloperonospora arabidopsidis (活体营养型卵菌), Trypan blue staining (台盼蓝染色), Haustoria quantification (吸器量化), Multilobed haustoria (多叶吸器), Plant-microbe interaction (植物微生物互作)

Background

Downy mildews are a group of phytopathogenic oomycetes characterized by branched sporangiophores that protrude through leaf stomata, giving the appearance of a white to grey “down” on the affected tissues (Agrios, 2005). Hundreds of Downy mildew species have been described on a wide variety of host plants including monocotyledon and dicotyledon crops (Callan and Carris, 2004). They are typically obligate biotrophs, meaning that they have not yet been successfully cultured outside the plant host with completion of their life cycle. A model system to study the molecular and genetic determinants underlying the downy mildew disease is the interaction between the host plant Arabidopsis thaliana and its oomycetal pathogen Hyaloperonospora arabidopsidis (Hpa). Downy mildew oomycetes typically form a structure called haustorium inside the plant host cell. The haustorium is surrounded by a plant cell membrane called extrahaustorial membrane (EHM) that prevents direct contact of the oomycete with the plant cytoplasm. While the hypothetical role of the haustorium in nutrient uptake from the plant host remains to be confirmed, it probably represents a platform for delivering oomycetal effectors to plants (Judelson and Ah-Fong, 2019) which enable the establishment and maintenance of a biotrophic interaction with the host. It is therefore not surprising that several proteins required for the resistance against Hpa are localized to the EHM (Wang et al., 2009; Caillaud et al., 2014). Despite the obvious relevance of the haustorium for the downy mildew interaction, surprisingly few studies evaluated the interaction by phenotyping the haustoria. A method for the inoculation of Arabidopsis by Hpa was described by Asai et al. (2015). Here we expanded this protocol resulting in a step-by-step guide for the study of this pathosystem in the lab. This includes details on how to sow and grow Arabidopsis, how to propagate Hpa, and specific details on how to systematically record structural features of this interaction with the Keyence VHX digital microscope, and quantify them with the image analysis software Fiji, here exemplified by the quantification of haustoria-shape variation. Our step-by-step guide for the microscopic analysis of the Hpa-Arabidopsis interaction should be easily adaptable for the use not only in other downy mildew interactions in host species other than Arabidopsis but also other hyphal pathogens other than oomycetes. The steps that most likely need attention when moving to other species combinations are highlighted in the protocol below.

Materials and Reagents

  1. Falcon® tube (50 ml) * 2 (Every similar-sized version will do)
  2. 2 ml Eppendorf® tubes (Every similar version will do)
  3. Miracloth® (8 cm x 8 cm) (Millipore, catalog number: 475855-1R )
  4. Arabidopsis thaliana Col-0, wildtype and shrk1 x shrk2 [shrk1 x shrk2 produces multilobed haustoria at higher frequency than the wildtype (Ried et al., 2019)]
  5. Hpa Noco2 (Col-0 is susceptible to the Hpa strain Noco2)
  6. Toothpick with pointed ends (see Figure 1)
  7. A white sheet of paper (A5 size, ISO 216)
  8. Several 6.5 cm x 7 cm x 7 cm pots with soil
  9. Plastic tray with a transparent lid [38 cm x 24 cm x 18 cm (height includes lid)]
    Note: The one we used is not produced anymore. Connex Indoor greenhouse, FLOR79045, is a similar version. Every similar sized box from a gardening supplier with a transparent lid should be suitable.
  10. Soil (Stender GmbH, A210)
  11. Fertilizer (WUXAL® Super 8-8-6)
  12. Counting chamber (Hemocytometer) (Marienfeld, catalog number: 0 640110 )
  13. Spray bottle
    Note: A small (20-30 ml volume) spray bottle from the cosmetic store which can produce a fine mist/aerosol is suitable for inoculation. Important! Use the same type for all your experiments and use the same distance and spray time to apply similar spore numbers from experiment to experiment. This may be critical for applying a similar infection pressure in each independent biological repeat.
  14. Ethanol 70% (for sterilization, ethanol of technical grade is sufficient)
  15. Sterile water
  16. Phenol (Roth, catalog number: 00 40.1 , any brand will do)
  17. Lactic acid (Roth, catalog number: 8460.1 , any brand will do)
  18. Trypan blue (Sigma, catalog number: T6146 , any brand will do)
  19. 35% (v/v) glycerol/water (Roth, catalog number: 7530.4 , any brand will do)
  20. 0.01% trypan blue solution (see Recipes)
  21. Saturated chloral hydrate solution (2.5 g/ml, see Recipes)

Equipment

  1. Scissors with a small and pointy front (Hammacher, HSB 544-09, any version similar to this should be suitable for cutting off infected leaves).
  2. Vortex (Vortex-Genie® 2, one with similar function will do)
  3. Growth chamber (Binder, model: KBWF 720 )
  4. Eppendorf ThermoMixer C (or other brand. Any temperature controlled Eppendorf® tube shaker with heating function will do)
  5. VHX-6000 digital microscope (Keyence, Osaka, Japan)
    Note: The advantage of using the VHX digital microscope is that it can record a large sample area by stitching together individual pictures in x, y and z directions. Any other bright field microscope with a 10x objective will be also suitable but perhaps less comfortable to use for this particular task.

Software

  1. Fiji (Schindelin et al., 2012, https://imagej.net/Fiji)
  2. Cell counter (A plugin for the Fiji software, Kurt De Vos, https://imagej.nih.gov/ij/plugins/cell-counter.html)

Procedure

  1. Growing of Arabidopsis
    1. Prepare Arabidopsis seedlings for the multilobed haustoria observation
      1. Stratify all the testing seeds at 4 °C overnight once after you harvest the seeds.
        Note: Stratification can synchronize the growing of the plants.
      2. Spread the amount of the seeds that you need onto a white paper (5-9 seeds for one pot) (Figure 1A).
      3. Moisture the tip of the toothpick by dipping it into water (Figure 1B).
      4. Distribute 5-9 seeds evenly in one 6.5 cm x 7 cm x 7 cm pot with the now sticky toothpick tip to pick up one seed each time (Figures 1C-1D).
        Notes:
        1. Don’t grow more than 12 plants in this pot size. A crowded pot will lead to an uneven infection because overlapping leaves will cover each other.
        2. Do not cover the seeds with the soil! Put the seeds directly on the surface of the soil.
      5. Grow plants at 22 °C, 16 h light (100 μmol/m2/s)/ 8 h dark, 60% relative humidity for 2 weeks. The plants were watered twice a week with the 1/1000 diluted fertilizer. Figure 1F shows 2-week-old plants grown under the conditions and pot size indicated.


      Figure 1. Procedure for growing and infecting the Arabidopsis. A. Spreading seeds evenly on a white sheet. B. Moisture the tip of the toothpick. C. Pick up one seed at once with a moisture tip. D. Put the seed on the surface of the soil. E. Pick up seeds by your index finger. F. 2-week-old Arabidopsis. G. The 2-week-old Arabidopsis’s leaves are evenly filled with the droplets. Scale bar = 0.8 cm. H. The 7 dpi Hpa infected Arabidopsis. The arrows show the first pair of leaves to collect. Scale bar = 0.8 cm.

    2. Prepare Arabidopsis seedlings for Hpa propagation
      1. Stratify all the testing seeds at 4 °C overnight once after you harvest the seeds.
      2. Spread the amount of the seeds that you need onto a white paper (about 50-100 seeds in one pot).
      3. Collect the seeds with your index finger and spread them onto the soil evenly like spreading salt on food (Figure 1E).
        Note: Do not cover the seeds with the soil! Put the seeds directly on the surface of the soil.
      4. Grow plants at 22 °C, 16 h light (100 μmol/m2/s)/8 h dark, 60% relative humidity for 2 weeks.

  2. Hpa propagation and preservation
    1. Hpa propagation
      1. Sterilize scissors with 70% ethanol.
      2. Clean and decontaminate the plastic tray and lid first with soap (detergent) then with 70% ethanol. Let them dry before further use.
      3. Place the pots which are prepared in Step A2 in the plastic tray and add 250 ml sterilized water in each tray to keep the humidity for the later step.
      4. Carefully open the tray of the 7 dpi Hpa infected plants and lift the cover slowly in a defined area for the Hpa infection. Reduce air movement around the plants to a minimum.
        Note: If you start the Hpa infection for the very first time in your lab, the Hpa source can be the pots of 7 dpi Arabidopsis or the freezed material (see Step B2) from the provider.
      5. Harvest the aerial parts of the infected plants covered with spores (Figure 2A) into a 50 ml Falcon® tube.
        Note: The infected Arabidopsis seedlings should be loosely placed in the tube to let the spores easily release to the sterilized water (see Figure 1B in Asai et al., 2015,). Usually, 2-3 full pots of the infected Arabidopsis are used.
      6. Add 15 ml of sterile water in the Falcon® tube and vortex it at the full speed (3,200 rpm) for 10 s.
      7. Filter the suspension using one layer of Miracloth.
      8. Measure the concentration of the conidiospores in the suspension by using the counting chamber (Hemocytometer). Adjust the concentration to 105 spores/ml water. If the spores are not enough, repeat Steps B1d-B1f for the rest of the materials.
      9. Spray the 2-week-old Arabidopsis with the filtered suspension until the leaf surface is evenly filled with the droplets (Figure 1G).
      10. Cover the plastic tray with the transparent lid and seal the openings with transparent tape to maintain high humidity (90%-100%).
      11. Incubate the infected plants in a growth chamber at 18 °C, 16 h light (80 μmol/m2/s*, 70 μmol/m2/s under the transparent lid)/8 h dark for 6 or 7 days.
        Notes: 
        1. *This is the actual measurement from the Binder chamber. The plants grow in the walk in chamber with the light intensity 100 μmol/m2/s, but the incubation has been done in the Binder chamber.
        2. For the propagation and the collection of the fresh conidiospores, Hpa should be maintained on Arabidopsis in a 6- or 7-day-regime. In other words, infect the plants every 6 or 7 days. Usually, Hpa will start to sporulate on 5 dpi. 6 dpi or 7 dpi are the better days for the conidiospores collection.
    2. Hpa preservation and regeneration
      1. Follow Steps B1a-B1e to collect the infected plants into a 50 ml Falcon® tube.
      2. Preserve the infected plants at -80 °C.
        Note: The Hpa conidiospores can be stored in this condition for at least 1 year or longer.
      3. For the regeneration, take out the preserved infected leaves from the -80 °C and put the tube on ice for 10 min.
      4. After 10 min, add 15 ml of sterile water in the Falcon® tube and vortex for 10 s at full speed.
      5. Filter the suspension using one layer of Miracloth.
      6. Spray the Arabidopsis (prepared from Step A2) with the filtered suspension until the leaf surface is evenly filled with the droplet (Figure 1G).
        Note: Since the viability of the frozen conidiospores will be lower, we don’t adjust (dilute) the concentration for the regeneration.

  3. Hpa inoculation and incubation
    1. Inoculation
      1. Randomly place different genotypes of Arabidopsis pots prepared from Step A1 in the tray.
      2. Infect the plants as in Step B1.
    2. Incubation
      Incubate the infected plants in a growth chamber at 18 °C, 16 h light (80 μmol/m2/s, 70 μmol/m2/s under the transparent lid)/ 8 h dark until the collection day.
      Note: Under these conditions, in the Arabidopsis thaliana Col-0 x Hpa Noco2 interaction, the multilobed haustoria start to accumulate on 5 dpi (Figure 3) and reach to a higher proportion at later infection stages (7 dpi-8 dpi) in both wild type and the shrk1 x shrk2 mutant.

  4. Staining of plant tissues for visualization of the pathogen
    1. Harvest one of the infected first pair of true leaves from each seedling (5-8 biological repeats for each genotype= 5-8 plants) (Figure 1H).
      Note: For other oomycete-plant pathosystems, the choice of leaf may vary. If nothing else matters, pick the youngest infected leaves as they are typically the smallest and easiest to stain.
    2. Incubate them in the trypan blue solution which cover the leaves in 2 ml Eppendorf® tubes at 95 °C for 3-10 min or until the tissues completely soaked up the solution and incubate them at room temperature overnight.
      Notes: 
      1. To avoid the splitting of the trypan blue while cooking the samples, don’t put trypan blue solution excess than 1 ml in the 2 ml Eppendorf®.
      2. Do not heat them longer than 30 min, the tissues will become too soft for further steps. However, the room temperature overnight process can be extended if the tissues are not stained enough. Roll the Eppendorf tube a bit to see whether the tissue is well soaked by the trypan blue. The ready tissues should be dark blue (Figure 2B).
      3. For the other oomycete pathosystems, heat and incubate the samples until the tissues completely soaked up the solution. The required incubation time may increase with leaf and cell wall thickness.
    3. Replace the solution with the saturated chloral hydrate solution and leave overnight until the tissues look clear (Figure 2C).
      Note: If the tissues are not yet clear, replace the chloral hydrate supernatant with a fresh aliquot and incubate until the tissues become clear.
    4. Replace the chloral hydrate with 35% glycerol.
      Note: The samples can be stored in 35% glycerol for long time (at least 3 months).
    5. Place the tissues on microscopy slides with 35% glycerol for further observation under the Keyence VHX digital microscope. Use a cover slip to prevent the sample from drying out.


      Figure 2. Hpa infected Arabidopsis and the trypan blue stained leaf. A. The non-infected mock leaf (left) and the 7 dpi Hpa infected leaf (right). Scale bar = 2 mm. B. The trypan blue-stained leaf before the de-staining process. Scale bar = 1 mm. C. The de-stained trypan blue-treated leaf. The hyphae can be clearly seen under the microscope or by the eyes. Scale bar = 1 mm.


      Figure 3. The Hpa haustoria in the Col-0 and the shrk1 x shrk2 leaf. The pictures were taken from the 5 dpi leaves by the 1,000x objective lens with the VHX digital microscope. The asterisks indicate the multilobed haustoria. Scale bars = 25 µm.
     
  5. An introduction of the Keyence VHX microscope and Fiji plugin of ImageJ for the quantification of structures of interest
    1. The using of the Keyence VHX digital microscope
      1. Turn on the microscope and follow the instruction of the software to position the sample in focus.
        Note: For the example showing below, we used 500x objective lens to visualize the samples.
      2. Use the “Stitching” function in the software to acquire pictures that contain larger areas.
      3. Use the “XY length” function in the “Stitching” window to set the area you want to include them in one picture (Figure 4, X direction 1,000 µm, Y direction 1,000 µm). The yellow square will also show you the area microscope will record this time.
        Note: X direction 1,000 µm and Y direction 1,000 µm is a suggested area size that works well for Arabidopsis leaves at the indicated dpi. You can change the area according to the biological system requirements, but always maintain one consistent size within the same experiment.


        Figure 4. The Stitching function in the VHX digital microscope software. The yellow square shows you the area microscope will record this time. The picture in the yellow square shows the position of the area which is displayed by the live imaging window now.

      4. Take pictures from 5 different and not overlapping areas from one leaf.
        Note: Take pictures continually by using the stitching function. It will show you the area you’ve just imaged. This guide should help to avoid overlaps between samples (Figure 5).


        Figure 5. The Stitching function helps to avoid overlaps between samples. The yellow square shows us the area just taken. This area should be excluded from additional stitchings from the same leaf to avoid overlapping samples.

    2. Quantification of multilobed and round-shaped haustoria by the Fiji plugin: cell counter
      Note: For a guide how to distinguish round-shaped (“normal”) and multi-lobed haustoria, see: Ried et al., 2019, Figure 2 or the figures taken by the VHX digital microscope in this protocol: Figure 3.
      1. Use the “cell counter” plugin in the Fiji package to count and record the haustoria shape.
      2. Open the picture in Fiji, click on the window of the picture and then start the “cell counter” plugin. Press “Initialize” to activate the picture you want to quantify (Figure 6).


        Figure 6. The plugin “cell counter” in the Fiji package. Initialization of the picture. Scale bar =100 μm.

      3. Define the name at the “Counters” (Figure 6 above, round-shaped (normal) haustoria; multilobed haustoria).
      4. Label them with a simple click beside the haustoria you want to label. Different types will then be labeled in different colors (Figure 7, normal haustoria in blue, multilobed haustoria in red).


        Figure 7. Use the plugin “cell counter” to label the haustoria. Label the haustoria by a simple click beside the haustoria you want to label. Scale bar = 100 μm.
        Note: Label only the haustoria which are in focus. Ignore the ones for which the shape is difficult to judge.

      5. You can change the labeling colors by clicking on the “options” (Figure 8).


        Figure 8. Change the labeling colors. Big ranges of the labeling colors can be chosen from the option window by right click on the colors. Scale bar = 100 μm.

      6. Pick up 2 or more branches from one picture to count the proportion of the multilobed haustoria (Figure 9). At least 10 branches in total should be counted in one biological repeat (one leaf).

      7. You can save the labels with the “Save Markers” function.


        Figure 9. The labeling of the Haustoria. A. Two branches from one picture. Scale bar = 100 µm. B and C. The magnification of the two branches. Scale bar = 50 µm. D. Haustoria labeled with different colors, green: normal haustoria, magenta: multilobed haustoria. Scale bar = 50 µm.

Recipes

  1. 0.01% trypan blue solution
    10 ml lactic acid*
    10 ml glycerol
    10 g phenol*
    10 ml sterile water
    10 mg trypan blue
    Note: Handle the materials labeled with asterisk cautiously in the chemical hood. Trypan blue is light sensitive. The solution should be stored in a brown bottle, it can be reused at least two times.
  2. Chloral hydrate solution (2.5 g/ml)
    Add 40 ml water to 100 g chloral hydrate in a 100 ml bottle
    Note: 100 g chloral hydrate will already occupy almost the entire space of a 100 ml bottle, therefore; don't put more than this amount in a 100 ml bottle.

Acknowledgments

The initial establishment of the Hpa x Arabidopsis pathosystem in the author’s laboratory was largely based on Hpa genotypes and advise given by the team of Jane Parker, while still working at The Sainsbury Laboratory in Norwich. The Steps B1 and D described in this protocol are similar to the one described by Asai et al. (2015). Procedures A-D were used as described in Ried et al. (2019). MP acknowledges funding by the Deutsche Forschungsgemeinschaft (DFG) and the European Research Council (ERC).

Competing interests

The authors have no competing interests to declare.

References

  1. Asai, S., Shirasu, K. and Jones, J. D. G. (2015). Hyaloperonospora arabidopsidis (Downy Mildew) Infection Assay in Arabidopsis. Bio-protocol 5(20): e1627.
  2. Caillaud, M. C., Wirthmueller, L., Sklenar, J., Findlay, K., Piquerez, S. J., Jones, A. M., Robatzek, S., Jones, J. D. and Faulkner, C. (2014). The plasmodesmal protein PDLP1 localises to haustoria-associated membranes during downy mildew infection and regulates callose deposition. PLoS Pathog 10(10): e1004496.
  3. Callan, B. and Carris, L. (2004). Biodiversity of Fungi: Inventory and Monitoring Methods. Elsevier Inc. pp: 113-114
  4. Judelson, H. S. and Ah-Fong, A. M. V. (2019). Exchanges at the Plant-Oomycete Interface That Influence Disease. Plant Physiol 179(4): 1198-1211. 
  5. Agrios, N. G. (2005). Plant Pathology. 5th edition. Elsevier Inc. pp. 427-433.
  6. Ried, M. K., Banhara, A. and Hwu, F. Y., Binder, A., Gust, A. A., Höfle, C., Hückelhoven, R., Nürnberger, T., Parniske, M. (2019). A set of Arabidopsis genes involved in the accommodation of the downy mildew pathogen Hyaloperonospora arabidopsidis. PLoS Pathog 15(7): e1007747.
  7. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J. Y., White, D. J., Hartenstein, V., Eliceiri, K., Tomancak, P. and Cardona, A. (2012). Fiji: an open-source platform for biological-image analysis. Nat Methods 9(7): 676-682. 
  8. Wang, W., Wen, Y., Berkey, R. and Xiao, S. (2009). Specific targeting of the Arabidopsis resistance protein RPW8.2 to the interfacial membrane encasing the fungal Haustorium renders broad-spectrum resistance to powdery mildew. Plant Cell 21(9): 2898-2913.

简介

[摘要] 寄主植物拟南芥(Arabidopsis thaliana,Arabidopsis)与卵菌的相互作用是研究专性生物营养性霜霉病相互作用的模型系统。对Hpa发育成功的评价通常是基于对叶片表面形成的生殖结构的量化,如孢子囊或其携带的分生孢子。然而,这种相互作用的结构基础在于植物组织,特别是在植物细胞内形成的吸器。因此,通过对植物组织内和细胞内菌丝形态和吸器形态的光镜观察,可以获得有关霜霉病相互作用的性能和相容性的有价值的附加信息。这里我们描述了一个可视化和量化植物内部形态表型的协议。虽然我们特别关注吸器形状变量的量化,但该协议可以很容易地适用于其他形态学特征的量化,如菌丝变形或卵原细胞频率。通过对已有的各种来源的协议进行归纳和提炼,我们组装了拟南芥Hpa生物测定的整个实验管道,为该系统在实验室的初步建立提供了实用的指导。该管道包括以下步骤:A)生长拟南芥;B)Hpa繁殖和菌株保持;C)Hpa接种和培养;D)植物组织染色以显示病原体;E)引入Keyence VHX显微镜和ImageJ的Fiji插件以量化感兴趣的结构。虽然这里描述的是拟南芥和Hpa,但B-E方案步骤应该很容易调整,以研究其他植物卵菌的病理系统。活体营养型卵菌(百帕)

[背景] 霜霉病是一组植物病原性卵菌,其特征是分枝的孢子囊团通过叶气孔突出,在受影响的组织上呈现白色到灰色“向下”的外观(Agrios,2005)。数以百计的霜霉病物种被描述在多种寄主植物上,包括单子叶和双子叶作物(Callan and Carris,2004)。它们是典型的专性生物群,这意味着它们还没有在植物寄主外成功培养完成其生命周期。研究霜霉病的分子和遗传决定因素的模型系统是:寄主植物拟南芥与其卵菌病原体霜霉病卵菌之间的相互作用,通常在寄主细胞内形成一种称为吸器的结构。吸器被称为吸器外膜(EHM)的植物细胞膜包围,防止卵菌与植物细胞质直接接触。虽然吸器在植物寄主吸收养分中的假设作用尚待证实,但它可能代表了一个向植物传递卵子效应器的平台(Judelson和Ah Fong,2019),该平台能够建立和维持与寄主的生物营养相互作用。因此,抗Hpa所需的几种蛋白质定位于EHM并不奇怪(Wang等人,2009;Caillaud等人,2014)。尽管吸器与霜霉病的相互作用有着明显的相关性,但令人惊讶的是,很少有研究通过吸器的表型来评估这种相互作用。Asai等人描述了用Hpa接种拟南芥的方法。(2015年)。在这里,我们扩展了这一方案,为实验室研究这一病理系统提供了一个循序渐进的指南,其中包括如何播种和种植拟南芥,如何传播Hpa,以及如何用Keyence VHX数字显微镜系统地记录这种相互作用的结构特征的具体细节,并用图像分析软件Fiji对其进行量化,以吸器形状变化的量化为例。我们的Hpa-拟南芥相互作用微观分析的分步指南应易于适应,不仅适用于除拟南芥以外的寄主物种中的其他霜霉病相互作用,而且也适用于除卵菌以外的其他菌丝病原体。在转移到其他物种组合时最可能需要注意的步骤在下面的协议中突出显示。活体营养型卵菌(人力资源部)。

关键字:拟南芥, 活体营养型卵菌, 台盼蓝染色, 吸器量化, 多叶吸器, 植物微生物互作

材料和试剂


 


1.     Falcon®试管(50毫升)*2(每个类似尺寸的型号都可以)


2.     2毫升Eppendorf®试管(每个类似版本都可以)


3.     米拉布(8 cm x 8 cm)(微孔,目录号:475855-1R)


4.     拟南芥Col-0,wildtype和shrk1 x shrk2[shrk1公司x shrk2产生多叶吸器的频率高于野生型(里德等人,2019年)]


5.     高功率放大器Noco2(Col-0对Hpa株Noco2敏感)


6.     尖头牙签(见图1)


7.     一张白纸(A5尺寸,ISO 216)


8.     几盆6.5厘米x 7厘米x 7厘米的土盆


9.     带透明盖的塑料托盘[38 cm x 24 cm x 18 cm(高度包括盖)]


注:我们用的那个已经不生产了。康奈克斯室内温室,FLOR79045,是一个类似的版本。园艺供应商提供的每一个类似大小的带透明盖子的盒子都应该是合适的。


10.  土壤(Stender GmbH,A210)


11.  肥料(WUXAL®Super 8-8-6)


12.  计数室(血细胞仪)(Marienfeld,目录号:0640110)


13.  喷雾瓶


注:化妆品商店提供的一个小(20-30毫升)喷雾瓶,可以产生喷雾/气溶胶适合接种。重要!在所有实验中使用相同的类型,并使用相同的距离和喷洒时间在每个实验中应用相似的孢子数。这对于在每个独立的生物重复中施加相似的感染压力可能是至关重要的。


14.  乙醇70%(用于灭菌,技术级乙醇即可)


15.  无菌水


16.  苯酚(Roth,目录号:0040.1,任何品牌都可以)


17.  乳酸(Roth,目录号:8460.1,任何品牌都可以)


18.  台盼蓝(Sigma,目录号:T6146,任何品牌都可以)


19.  35%(v/v)甘油/水(Roth,目录号:7530.4,任何品牌均可)


20.  0.01%台盼蓝溶液(见配方)


21.  水合氯醛饱和溶液(2.5g/ml,见配方)


 


设备


 


1.     小而尖的剪刀(Hammacher,HSB 544-09,任何类似的版本都应该适合剪下被感染的叶子)。


2.     漩涡(漩涡精灵2,功能相似的一个)


3.     生长室(粘合剂,型号:KBWF 720)


4.     Eppendorf热混合器C(或其他品牌。任何带加热功能的温控Eppendorf®振动筛都可以)


5.     VHX-6000数字显微镜(日本大阪凯恩斯)


注:使用VHX数字的优势显微镜是通过将x、y和z方向上的个别图片拼接在一起来记录大面积的样品s公司. 任何其他带有10倍物镜的明亮视野显微镜也适用于此项特殊任务,但可能不太舒适。


 


软件


 


1.     斐济(Schindelin等人,2012,https://imagej.net/斐济)


2.     Cell counter(斐济软件的插件,Kurt De Vos,https://imagej.nih.gov/ij/plugins/cell-counter.html)


 


程序


 


A、 拟南芥的生长


1.     拟南芥幼苗的多叶吸器观察


a、 收获种子后,将所有测试种子在4°C下隔夜分层。


注:分层可以使植物生长同步。


b、 把你需要的种子撒在白纸上(一盆5-9粒)(图1A)。


c、 将牙签的顶端浸入水中使其湿润(图1B)。


d、 将5-9粒种子均匀地分布在一个6.5 cm x 7 x 7 cm的罐子中,罐子上有粘性的牙签尖,以便每次都能捡到一粒种子(图1C-1D)。


笔记:


一。不要在这个花盆里种超过12株植物。拥挤的花盆会导致不均匀的感染,因为重叠的叶子会互相覆盖。


二。不要用土覆盖种子!把种子直接放在土壤表面。


e、 在22°C,16h光照(100mol/m2/s)/8h黑暗,60%相对湿度下生长2周。这些植物每周用1/1000稀释肥料浇水两次。图1F显示了在所示条件和盆栽大小下生长的2周龄植物。μ


 


 


图1。拟南芥生长和感染的过程。 A、 把种子均匀地撒在白纸上。B、 把牙签尖弄湿。C、 用湿尖一次捡起一粒种子。D、 把种子放在土壤表面。E、 用食指捡起种子。F、 2周大的拟南芥。G、 两周大的拟南芥的叶子上均匀地充满了水滴。比例尺=0.8 cm。H、 7dpi-Hpa感染拟南芥。箭头显示要收集的第一对树叶。比例尺=0.8 cm。s公司


 


2.     拟南芥Hpa育苗的研究


a、 收获种子后,将所有测试种子在4°C下隔夜分层。


b、 把你需要的种子撒在白纸上(一盆大约50-100粒种子)。


c、 用食指把种子收集起来,像在食物上撒盐一样均匀地撒在土壤上(图1E)。


注意:不要用泥土覆盖种子!把种子直接放在土壤表面。


d、 在22°C,16h光照(100μmol/m2/s)/8h黑暗,60%相对湿度下生长2周。


 


 


B、 繁殖与保存高功率放大器


1.     高功率放大器传播


a、 用70%乙醇消毒剪刀。


b、 先用肥皂(洗涤剂)清洗塑料托盘和盖子,然后用70%乙醇清洗。在进一步使用前让它们干燥。


c、 将步骤A2中准备好的锅放入塑料托盘中,并在每个托盘中加入250毫升消毒水,以保持湿度,以备后续步骤使用。


d、 小心打开7 dpi Hpa感染植物的托盘,在Hpa感染的指定区域缓慢提起盖子。尽量减少植物周围的空气流动。


注:如果你是第一次在实验室感染Hpa,Hpa的来源可以是7 dpi拟南芥或供应商提供的冷冻材料(见步骤B2)。


e、 将被孢子覆盖的受感染植物的地上部分(图2A)采集到50毫升的Falcon®试管中。


注:受感染的拟南芥幼苗应松散地放在试管中,以使孢子容易释放到灭菌水中(见Asai等人,2015年,图1B)。通常使用2-3整盆受感染的拟南芥。


f、 在Falcon®试管中加入15毫升无菌水,以全速(3200转/分)旋转10秒。


g、 用一层薄纱过滤悬浮液。


h、 用计数室(血细胞仪)测定悬浮液中分生孢子的浓度。将浓度调整为105孢子/ml水。如果孢子不够,对其余材料重复步骤B1d-B1f。


i、 用过滤后的悬浮液喷洒2周大的拟南芥,直到叶面均匀地充满水滴(图1G)。


j、 用透明盖盖住塑料托盘,并用透明胶带密封开口,以保持高湿度(90%-100%)。


k、 将受感染的植物在18°C、16h光照(80μmol/m2/s*、70μmol/m2/s透明盖下)的生长室内培养6或7天。


笔记:


答。*这是粘合剂室的实际测量值。植物生长在光照强度为100的步入式室内μmol/m2/s,但已在粘合剂室中进行培养。


乙。为了繁殖和收集新鲜的分生孢子,在拟南芥上应保持6-7天的Hpa。换言之,每6或7天感染一次。通常,Hpa在5dpi时开始孢子形成。6dpi或7dpi是孢子收集的较好时期。


2.     高功率放大器保存与再生


a、 按照步骤B1a-B1e将受感染的植物收集到50毫升的Falcon®试管中。


b、 将受感染的植物保存在-80°C下。


注:Hpa分生孢子可在此条件下保存至少1年或更长时间。


c、 为了再生,从-80°c温度下取出保存好的感染叶片,将试管放在冰上10分钟。


d、 10分钟后,在Falcon®试管中加入15毫升无菌水,全速涡流10秒。


e、 用一层薄纱过滤悬浮液。


f、 用过滤后的悬浮液喷洒拟南芥(从步骤A2制备),直到叶表面均匀地充满液滴(图1G)。


注:由于冷冻分生孢子的活力较低,我们不调整(稀释)再生浓度。


 


C、 接种和培养高功率放大器


1.     接种


a、 将从步骤A1制备的不同基因型的拟南芥盆随机放置在托盘中。


b、 按照步骤B1感染植物。


2.     孵化


将受感染的植物在18°C、16h光照(80μmol/m2/s,70μmol/m2/s,透明盖下)的生长室内培养/8h,直到收集日。


注:在这些条件下,拟南芥Col-0x Hpa-Noco2相互作用中,多叶吸器开始在5dpi上积累(图3)野生型和shrk1×shrk2突变体在感染后期(7dpi-8dpi)均达到较高比例。


 


D、 植物组织染色观察病原菌


1.     从每株幼苗中采集一对受感染的第一对真叶(每个基因型5-8个生物学重复=5-8株)(图1H)。


注:对于其他卵菌科植物的病理系统,叶子的选择可能会有所不同。如果没有其他问题,选择最年轻的感染叶,因为它们通常是最小和最容易染色。


2.     将它们在台盼蓝溶液中培养3-10分钟,台盼蓝溶液在95°C的2毫升Eppendorf®试管中覆盖叶子,或直到组织完全吸收溶液并在室温下培养过夜。


笔记:


答。为避免烹调样品时台盼蓝的分裂,不要将超过1毫升的台盼蓝溶液放入2毫升Eppendorf®中。


乙。加热时间不要超过30分钟,否则组织将变得太软,无法继续进行下一步。但是,如果组织染色不够,室温过夜过程可以延长。将Eppendorf试管稍微转动一点,看看组织是否被台盼蓝浸透。准备好的组织应该是深蓝色(图2B)。


c。对于其他卵菌病理系统,加热并培养样品,直到组织完全吸收溶液。所需的培养时间可能随着叶片和细胞壁厚度的增加而增加。


3.     用饱和水合氯醛溶液代替溶液,过夜,直到组织看起来清晰为止(图2C)。


注意:如果组织还不清楚,用新鲜的等分液替换水合氯醛上清液,并孵化,直到组织变得清楚。


4.     用35%甘油代替水合氯醛。


注:样品可在35%甘油中长期保存(至少3个月)。


5.     将组织置于35%甘油的显微镜载玻片上,在Keyence VHX数字显微镜下进一步观察。使用盖玻片防止样品干燥。


 


 


图2。Hpa感染拟南芥和台盼蓝染色叶片。 A、 未感染的模拟叶(左)和7dpi-Hpa感染的叶(右)。比例尺=2 mm。B、 台盼蓝在去染色前染色。比例尺=1 mm。C、 去染色的台盼蓝处理过的叶子。菌丝在显微镜下或肉眼都能清楚地看到。比例尺=1 mm。


 


 


图3。Col-0和shrk1 x shrk2叶中的Hpa吸器。 用VHX数码显微镜,用1000x物镜从5dpi叶片上拍摄。星号表示多叶吸器。比例尺=25微米。


 


E、 介绍用于感兴趣结构量化的Keyence VHX显微镜和ImageJ的Fiji插件


1.     Keyence-VHX数字显微镜的应用


a、 打开显微镜,按照软件的说明将样品对焦。


注意:对于下面显示的示例,我们使用丁用500倍物镜观察样品。


b、 使用软件中的“缝合”功能获取包含较大区域的图片。


c、 使用“缝合”窗口中的“XY长度”功能设置要在一张图片中包括它们的区域(图4,X方向1000μm,Y方向1000μm)。黄色的正方形也会显示区域显微镜这次会记录。


注:X方向1000μm和Y方向1000μm是建议的面积大小,适用于指示dpi处的拟南芥叶片。你可以根据生物系统的要求改变面积,但在同一个实验中要始终保持一个一致的大小。


 


 


图4。VHX数字显微镜软件中的拼接功能。 黄色的方块向你展示了这一次显微镜将记录的区域。黄色方块中的图片显示了实时图像窗口显示的区域的位置。


 


d、 从一片叶子上的5个不同且不重叠的区域拍照。


注意:使用缝合功能连续拍照。它会显示你刚刚拍摄的区域。本指南有助于避免样本之间的重叠(图5)。


 


 


图5。缝合功能有助于避免样本之间的重叠。 黄色的正方形向我们展示了刚才的面积。该区域应排除在同一片叶子的额外缝合之外,以避免重叠样本。


 


2.     Fiji插件对多叶圆形吸器的定量:细胞计数器


注:有关如何区分圆形吸器(“正常吸器”)和多叶吸器的指南,请参见:Ried等人,2019年,图2或本协议中由VHX数字显微镜拍摄的图像:图3。


a、 使用Fiji包中的“cell counter”插件计算并记录吸器形状。


b、 在斐济打开图片,点击图片窗口,然后启动“细胞计数器”插件。按“Initialize”激活要量化的图片(图6)。


 


 


图6。斐济包中的插件“cell counter”。 初始化图片。比例尺=100μm。


 


c、 在“计数器”处定义名称(上图6,圆形(正常)吸器;多叶吸器)。


d、 只需在吸器旁边简单地单击即可标记它们。不同类型的吸器会被标记成不同的颜色(图7,正常吸器为蓝色,多叶吸器为红色)。


 


 


图7。使用插件“细胞计数器”标记吸器。 只需单击要标记吸器的旁边,即可标记吸器。比例尺=100μm。


注意:请仅标记焦点所在的吸器。忽略那些形状难以判断的。


 


e、 您可以通过点击“选项”(图8)来更改标签颜色。


 


 


图8。更改标签颜色。 通过右键单击颜色,可以从选项窗口中选择大范围的标签颜色。比例尺=100μm。


 


f、 从一张图片中选取2个或更多分支来计算多叶吸器的比例(图9)。一个生物重复(一片叶子)中至少有10个分枝。


g、 可以使用“保存标记”功能保存标签。


 


 


图9。吸器的标签。 A、 一幅画上的两个树枝。比例尺=100μm。B和C。两个分支的放大倍数。比例尺=50μm.D.吸器用不同颜色标记,绿色:正常吸器,洋红:多叶吸器。比例尺=50微米。


食谱


 


1.     0.01%台盼蓝溶液10毫升乳酸*10毫升甘油10克苯酚*10毫升无菌水10毫克台盼蓝


注意:在化学罩内小心处理标有星号的材料。台盼蓝对光敏感。溶液应贮存在棕色瓶中,至少可重复使用两次。


2.     水合氯醛溶液(2.5 g/ml)


将40毫升水加入100克水合氯醛的100毫升瓶中


注:100克水合氯醛几乎已经占据了100毫升瓶子的全部空间,因此,不要在100毫升瓶子中放入超过此量的水。


 


致谢


 


作者实验室最初建立的Hpa x拟南芥病理系统主要是基于Hpa基因型,并由简·帕克团队提供建议,同时还在诺维奇的塞恩斯伯里实验室工作。本协议中描述的步骤B1和D与Asai等人描述的步骤类似。(2015年)。程序A-D如Ried等人所述。(2019年)。国会议员承认德国科学院(DFG)和欧洲研究理事会(ERC)的资助。


 


相互竞争的利益


 


作者没有相互竞争的利益要申报。


 


工具书类


 


1.     Asai,S.,Shirasu,K.和Jones,J.D.G.(2015年)。拟南芥霜霉病感染试验。生物协议5(20):e1627。


2.     Caillaud,M.C.,Wirthmueller,L.,Sklenar,J.,Findlay,K.,Piquerez,S.J.,Jones,A.M.,Robatzek,S.,Jones,J.D.和Faulkner,C.(2014年)。胞间连丝蛋白PDLP1在霜霉病感染期间定位于吸器相关膜并调节胼胝质沉积。公共科学图书馆病理学10(10):e1004496。


3.     Callan,B.和Carris,L.(2004)。真菌生物多样性:调查和监测方法。爱思唯尔公司,pp:113-114


4.     Judelson,H.S.和Ah Fong,A.M.V.(2019年)。影响疾病的植物卵菌界面的交换。植物生理学179(4):1198-1211。


5.     Agrios,N.G.(2005年)。植物病理学。第五版。爱思唯尔公司,第427-433页。


6.     Ried,M.K.,Banhara,A.和Hwu,F.Y.,Binder,A.,Gust,A.A.,Hófle,C.,Hückelhoven,R.,Nürnberger,T.,Parniske,M.(2019年)。一组拟南芥基因参与了霜霉病病原菌拟南芥的调节。公共科学图书馆病理学15(7):e1007747。


7.     Schindelin,J.,Arganda Carreras,I.,Frise,E.,Kaynig,V.,Longair,M.,Pietzsch,T.,Preibisch,S.,Rueden,C.,Saalfeld,S.,Schmid,B.,Tinevez,J.Y.,White,D.J.,Hartenstein,V.,Eliceri,K.,Tomancak,P.和Cardona,A.(2012年)。斐济:一个开放源码的生物图像分析平台。Nat方法9(7):676-682。


8.     Wang,W.,Wen,Y.,Berkey,R.和Xiao,S.(2009年)。拟南芥抗性蛋白RPW8.2特异性靶向真菌吸器周围的界面膜,使其具有广谱抗白粉病能力。植物细胞21(9):2898-2913。


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引用:Hwu, F. and Parniske, M. (2020). Maintenance and Quantitative Phenotyping of the Oomycete-plant Model Pathosystem Hyaloperonospora arabidopsidisArabidopsis. Bio-protocol 10(12): e3661. DOI: 10.21769/BioProtoc.3661.
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