Isolation of Powdery Mildew Haustoria from Infected Barley
感染大麦中白粉病吸器的分离提取   

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Thomas Torode Thomas Torode
Hailong Guo Hailong Guo
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Methods
Aug 2011

 

Abstract

Blumeria graminis is a fungus that causes powdery mildews on grasses, such as barley. Investigations of this pathogen present many challenges due to its obligate biotrophic nature. This means that the fungus can only grow in the presence of a living host plant. B. graminis forms epiphytic mycelia on the plant surface and feeding organs (haustoria) inside the epidermal cells of the host plant. Therefore, it is difficult to separate the fungus from plant tissues. This protocol shows how to obtain different fungal structures from powdery mildew infected barley leaves. The epiphytic mycelia including conidia and conidiophores can be separated after immersing the infected leaves into 5% cellulose acetate dissolved in acetone, and peeling off the cellulose acetate membrane. Then, the haustoria are isolated from dissected epidermis after cellulase degradation of plant cell walls. The isolated haustoria remain intact with few plant impurities. The haustoria may be visualized by epifluorescence microscopy after staining with the chitin-specific dye WGA-Alexa Fluor 488. Finally, dissected material can be either processed immediately or kept at -80 °C for long-term storage for studies on gene expression and protein identification, for example by mass spectrometry.

Keywords: Powdery mildew (白粉病), Barley (大麦), Epiphytic (附生的), Epidermis (表皮), Haustoria isolation (吸器提取), Cellulase (纤维素酶), WGA-AF 488 (WGA-AF 488)

Background

Powdery mildew of cereals, caused by Blumeria graminis, leads to significant yield loss (Spanu et al., 2010). The barley powdery mildew fungus B. graminis f. sp. hordei, is economically important, and one of the best studied powdery mildews (Both et al., 2005a; Bindschedler et al., 2009). This fungus is an obligate biotroph, which means it can only infect and propagate on living barley plants (Pedersen et al., 2012). Its asexual life cycle involves an intimate relationship with the host plant. Airborne conidia germinate on the host producing first a primary and then a secondary germ tube. The secondary germ tube develops an appressorium, from which a hyphal peg penetrates into an epidermal cell. Inside the cell, a penetration peg enlarges and forms a multidigitate feeding structure: the haustorium. This is surrounded by a plant-derived extrahaustorial membrane (Both et al., 2005b; Bindschedler et al., 2016).

Several studies of B. graminis f. sp. hordei have characterized gene expression profiles and proteomics of this fungus (Bindschedler et al., 2011; Pennington et al., 2016). However, the obligate biotrophic nature of B. graminis poses exceptional challenges to the investigations of this fungus, especially the studies of haustoria. The intracellular haustorium and the extrahaustorial membrane constitute a special compartment, which is functionally essential for the interaction with the host (Wang et al., 2009; Pliego et al., 2013). Investigating haustoria is critical for further understanding the mechanism of nutrient uptake into the pathogen as well as plant-pathogen recognition. Therefore, obtaining purified haustoria from infected plants has the potential of contributing significantly to these studies.

Previously, the procedure for isolating haustoria from obligate biotrophs employed affinity chromatography on rust fungi (Catanzariti et al., 2006; Garnica et al., 2014). During the 1990s, several centrifugation-based methods were introduced to isolate powdery mildew haustoria from pea plants (Mackie et al., 1991; Testut et al., 1999). This method was then applied to powdery mildews of other plants, including Arabidopsis (Wang et al., 2009; Micali et al., 2011). More recently, a filtration and gradient centrifugation-based method was described to isolate powdery mildew haustoria from barley (Godfrey et al., 2009). The hallmark of these published methods is that they are relatively laborious and require several different kinds of isolation buffers, different sizes of steel meshes and multiple centrifugation steps. Besides, the isolation procedure was required to be performed on ice. In this protocol, we developed a simpler way to isolate B. graminis haustoria from infected barley leaves. Releasing of haustoria from plant cells is achieved by enzymatically degrading the epidermal cell walls, and the purification of haustoria by filtration through a nylon mesh. This method requires digestion of the host plant cell walls, a single filtration and one centrifugation step.

Materials and Reagents

  1. Pipette tips
  2. Dedicated enclosed transparent container
  3. Razor blade
  4. Parafilm
  5. Pasteur pipettes
  6. 15 ml/50 ml polypropylene tubes
  7. Liquid nitrogen container
  8. Microscope glass slide
  9. Cover glass
  10. Barley (Hordeum vulgare) cv. Golden Promise seedlings, barley seeds were sown in John Innes 1 compost mixed with vermiculite (4:1) in 12 x 12 cm pots
  11. Liquid nitrogen
  12. Cellulose acetate
  13. Anhydrous acetone
  14. MES hydrate powder
  15. HCl
  16. WGA, Alexa Fluor 488 conjugate ( Thermo Fisher Scientific, catalog number: W11261 )
    Note: This dye exhibits the bright, green fluorescence of the Alexa Fluor® 488 dye (excitation/emission maxima ~495/519 nm).
  17. Cellulase Onozuka R-10 (Duchefa Biochemie, catalog number: C8001)
    Note: “Cellulase Onozuka R-10” from Trichoderma viride.1 unit (U) of Cellulase will release 1.0 μmole of glucose from carboxymethyl cellulose.
  18. NaCl
  19. KCl
  20. Na2HPO4
  21. KH2PO4
  22. PBS buffer (see Recipe 1)

Equipment

  1. Forceps
  2. Pipettes
  3. Scissor
  4. Tweezers
  5. 70 μm nylon mesh sieve
  6. Incubator
  7. Centrifuge
  8. GFP filter
  9. Epifluorescence microscope
  10. -80 °C freezer

Procedure

  1. Dissection of infected barley leaves
    1. Grow barley (Hordeum vulgare) cv. Golden Promise seedlings in a 16-h light/8-h dark cycle at 20 °C. It is essential to keep the plants well-watered, as drought stress prevents effective dissection of the epidermis.
    2. Maintain the powdery mildew fungus (B. graminis f. sp. hordei) on living barley plants in a dedicated enclosed transparent container protected from air currents that may displace the conidia. Remove old conidia by shaking the inoculum pots one day before infecting healthy plants.
    3. Inoculate seven-day barley seedlings. At this stage, the primary leaves are fully extended. The inoculation B. graminis is carried out by shaking heavily infected plants over the uninfected barley. The plants will be ready for dissection seven days after inoculation.
      Note: It is highly recommended that a tight-fitting face mask be used when carrying out this procedure to prevent conidia from entering the operator’s airways.
    4. Prepare scissor, tweezers, razor blade, Parafilm, Pasteur pipettes, 15 ml/50 ml polypropylene tubes and liquid nitrogen. Place two 50 ml tubes in a liquid nitrogen container and fill with liquid nitrogen.
    5. Dissolve 5 g of cellulose acetate in 100 ml anhydrous acetone. Pour the solution into a 15 ml polypropylene tube.
    6. Dissolve 0.2 g of MES hydrate powder in 100 ml H2O to make 10 mM MES buffer. Adjust the pH to 5.3 with HCl. Pour 20 ml of MES buffer into a 50 ml tube at room temperature.
    7. Cut one infected primary leaf and immerse it in 5% cellulose acetate acetone, holding it at the leaf base. Carefully take out the leaf and wait for a few seconds above the mouth of the tube to remove any cellulose acetate acetone droplets accumulating at the leaf tip. Then place the leaf onto two Pasteur pipettes.
    8. Repeat the above step five times. Let the acetone evaporate from the leaf surface for 5-8 min.
      Note: This step is critical: the cellulose acetate membrane needs to be dry enough to allow peeling; however, if the leaves are left too long they dehydrate and wilt rendering the dissection of the epidermis impossible.
    9. As the acetone evaporates, the epiphytic mycelia-containing cellulose acetate layer will harden and begin to peel away from the rest of the leaf. Now peel off the cellulose acetate membrane with the epiphytic fungal structures embedded in it. Keep the leaves stripped of epiphytic structures on water for 5 min to ensure full hydration.
    10. Following this, turn the leaf so the adaxial surface faces upwards. Press a razor gently near the tip of the leaf, carefully rock the blade back and forward.
      Note: This step is challenging and requires some trial and error to gain sufficient experience to achieve correct dissection. The aim is to cut through the upper, adaxial epidermis and part of the mesophyll, while retaining the lower, abaxial epidermis intact.
    11. Turn the leaf over to let the abaxial side facing upwards. While gripping the leaf at its tip (this forms a ‘tab’ which can be used to pull the epidermis) and holding the section of the leaf below the cut steady, pull back the abaxial epidermis which contains fungal haustoria.
      Note: Free the epidermal layer by pushing away other layers with forceps, if needed.
    12. Once free, carefully peel the abaxial epidermis in a continuous smooth motion.
    13. Once complete, take away the tip of the leaf (the ‘tab’) by cutting the epidermis and removing it with the tweezers.
      Note: To isolate haustoria from epidermis, skip to Step B1.
    14. Snap freeze peeled epiphytic and epidermal materials separately in two 50 ml polypropylene tubes in liquid nitrogen for future use.
    15. Store at -80 °C till needed.

  2. Haustoria isolation
    1. Float peeled epidermis in 15 ml 10 mM MES hydrate buffer (pH 5.3) in a 50 ml tube.
    2. Once sufficient epidermis strips are collected (we used one pot in Video 1), add 200 mg of Cellulase R-10 into 10 ml MES buffer in a new 50 ml tube. Mix thoroughly, so the final concentration of cellulase is 2% (w/v).
    3. Carefully transfer all dissected epidermis with tweezers into the new mixture.
    4. Incubate the suspension at 28 °C for two hours with gentle shaking (e.g., 80 rpm on a rotating platform). During incubation, the cellulase will lyse the barley epidermal cells and release haustoria into the incubation buffer.
    5. After incubation, place a 70 μm nylon mesh sieve over a 50 ml tube. Slowly pour the buffer through the filter while using forceps as a barrier to retain larger pieces of undigested plant material.
    6. Once all the buffers are collected, centrifuge in a swing out rotor at 3,270 x g for 10 min, at 4 °C.
    7. After centrifugation, the pellet should contain haustoria.
    8. Pour off the supernatant and retain the pelleted haustoria.
      Note: After this step, the haustoria can either be kept at -80 °C for long-term storage or stained immediately to visualize (continue with Procedure C).

  3. WGA-Alexa Fluor 488 staining (optional for quality control)
    1. Gently resuspend the haustorial pellet in 300 μl of PBS buffer (pH 7.4) (Recipe 1).
    2. Add WGA-Alexa Fluor 488 into the mixture to a final concentration of 10 μg/ml.
    3. Mix well by inverting the tube several times.
    4. Load the mixture onto a microscope glass slide and carefully place a cover glass on top.
    5. Visualize under the GFP filter with an epifluorescence microscope.

      Video 1. Visualization of the protocol as a video recording of the procedures

Data analysis

Representative results: The protocol described above provides an efficient enzymatic way to obtain B. graminis haustoria from the host plant, barley. All materials obtained using this protocol, including epiphytic mycelia-contained acetate stripes, haustoria-present epidermal peels and purified haustoria, can be stored at -80 °C for long-term storage till needed for further analysis. One pot of heavily infected barley (roughly 80 leaves) yields approximately 1.5 g of epiphytic material (N.B. this includes the cellulose acetate film, which constitutes the major part of this fraction, by weight), 0.3 g of epidermis and 3-4 x 105 haustoria (the actual number of haustoria obtained will depend strongly on the extent to which the leaves were infected). The quality of isolated haustoria can be observed by epifluorescence microscopy after staining by WGA-Alexa Fluor 488. Most of the isolated haustoria are highly branched and intact (Figure 1A). Sometimes the separated haustorium is still surrounded by the plant-derived extrahaustorial membrane, as shown in Figure 1B (arrow). Plant fragments which are small enough can also pass through the 70 μm nylon mesh sieve, so limited amount of impurities are present in pelleted haustoria complex, for example chloroplast (Figure 2, arrow).


Figure 1. Isolated haustoria visualized after staining with WGA-Alexa Fluor 488. A. Stained haustoria (fluorescent green) visualized by epifluorescence microscopy and observed through a “GFP” filter. Multiple intact and mature haustoria. Scale bar = 40 μm. B. This shows both the multidigitated haustorium and a perihaustorial membrane surrounding the haustorium (arrow). Scale bars = 10 μm.


Figure 2. An isolated haustorium with some impurities. The pellet formed after filtration and centrifugation not only consists of haustoria, but also some plant debris, for example chloroplasts (autofluorescent red, arrow). These impurities could be chloroplasts and cell wall fragments. Scale bar = 10 μm.

  We tested several options for overall digestion time in cellulase: 1 h, 2 h, 3 h and overnight. Overnight incubation yielded the highest number of haustoria: approximately 5 x 105 (this was considered the maximum amount of haustoria we can achieve), but also more fragments of plant material which reduced the purity of isolated haustoria. One-hour incubation only released less than 30% of the maximum obtained after overnight digestion. Digestions of 2 h and 3 h incubation yielded 3-4 x 105 haustoria, which accounts for > 80% of the maximum.

Discussion: Powdery mildew fungi are challenging to study because they have an absolute requirement for a host plant to grow and develop. This is especially true of the haustoria, the feeding structures which develop inside the host plant epidermal cells (Catanzariti et al., 2006; Micali et al., 2011). Several affinity isolation methods of powdery mildew haustoria have been previously published, including one modified method on B. graminis (Mackie et al., 1991; Micali et al., 2008). However, the method demonstrated for B. graminis, which combined gradient centrifugation and multiple filtrations, produce a pellet with 80% haustoria; the rest were reported to be cell wall fragments and chloroplasts (Godfrey et al., 2009). Isolating protoplast from different plants in previous studies is mostly achieved by a combination of enzymes, including cellulose (Sun et al., 2013; Wu et al., 2017). To obtain relatively pure and intact haustoria from barley powdery mildew, we developed a rapid and effective method which relies on the dissection of infected epidermis from the rest of the leaf followed by degradation of the plant cell walls and lysis of the epidermal cells using cellulase.  
  Maximizing the number of haustoria present in infected leaves is important when developing the protocol. Barley cv. Golden Promise was chosen to maintain the fungi due to its high susceptibility to powdery mildew. An appropriate number of seeds were sown to ensure there are enough plants while the density of the seedling is not too high to interfere with a saturating inoculation of the primary leaves. In these studies, we used approximately 3 g of seeds for a 10 cm-diameter pot. In order to ensure a high infection rate, conidia that are older than one day need to be removed from the plants used as a source of the inoculum. In this way, we ensure that only fresh conidia (< 1 day old) are used, leading to high percentage of successful germination, penetration and finally haustoria formation. The effectiveness of epidermis dissection is critical for the success of haustoria isolation. The young leaves are easier to dissect: there is a trade-off between more advanced stages of infection (yielding more haustoria) and younger leaves (yielding more intact epidermis). In practice, we have found that using plants 7 days post inoculation (dpi) as an ideal compromise. In addition, the epidermis of the abaxial side of primary leaf is the easiest to dissect compared to either the adaxial epidermis, or to the epidermis of other leaves. We therefore routinely dissect only the abaxial epidermis of the primary leaves.  
  To optimize the condition of haustoria isolation, several different incubation times and enzyme concentrations have been tested. Increasing concentration of cellulase (higher than 2.5%) and a combination of various enzymes (cellulase and macerozyme) have been proven effective in degrading cell walls, however, they were found to be costly and led to low viability of isolated structures (Wu et al., 2017). Accordingly, we used 2% of cellulase to break down the cell walls for releasing haustoria. The time of incubation in cellulase was critical to obtain high yield of isolated haustoria and low contamination from plant derived structures. After comparing different incubation times, we suggest that 2 h is a suitable compromise, for most purposes.  
  This protocol utilized WGA-AF 488 to stain isolated haustoria rather than the traditional method of using Coomassie blue. The lectin moiety binds to fungal chitin and rendered the fungal structures visible as green fluorescent structures due to the Alexa 488 fluorophore clearly distinguishable from plant derived material. If required, it would be possible to visualize the contaminating plant cell wall residues by staining with Calcofluor White.  
  We believe this protocol may be applied to other powdery mildews with suitable modifications.

Recipes

  1. 1x PBS buffer
    8 g NaCl
    0.2 g KCl
    1.44 g Na2HPO4
    0.24 g KH2PO4
    Dissolved in 800 ml distilled water
    Adjust the pH to 7.4 with HCl
    Add distilled water to a total volume of 1 L

Acknowledgments

LL was funded by the China Scholarship Council (CSC) studentship, and BC was funded by The Gregory Trust (Imperial College London).

Competing interests

The authors declare that there are no competing interests in the work described here.

References

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简介

对这种病原体的调查是由于造成大麦等禾本科植物的霉菌。对许多病原体生物制剂的研究归因于生物医学性质。这意味着只能体验这种病活体寄主植物。 B.graminis 在寄主植物的硬膜外细胞内的植物表面和饲养器官(吸器)上形成附生菌丝体。难以将真菌与植物组织分离。 (5)醋酸纤维素断开纤维素协议协议协议协议显示ob fun协议有趣的epi epi epi epi epi epi epi epi epi epi epi epi协议epi协议epi协议植物细胞纤维素酶降解后,解剖表皮中分离出吸器,分离出的吸器完好无损,含有多种植物杂质。用几丁质特异性染料WGA-Alexa Fluor 488染色后进行落射荧光显微镜检查。最后,解剖材料可立即加工或在-80°C下长期保存,用于基因表达和鉴定研究质谱。
【背景】谷类白粉病,致白粉菌,导致显著的产量损失(Spanu 等人,2010)。该大麦白粉病菌 B.菌< sp。 hordei ,在经济上很重要,是研究得最好的白粉病之一( et al。,2005a; Bindschedler et al。 ,2009)。这种真菌是一种专性biotroph,这意味着它可以只感染和对活大麦植物繁殖(Pedersen的等人,2012)。它的无性生命周期涉及的紧密空气中的分生孢子在宿主上萌发,首先产生一个初级胚芽,然后是一个次级胚芽管。二级胚芽管形成一个附着物,一个菌丝钉穿过附着物进入上皮细胞。 。并形成多指馈送结构:..吸器这是由植物来源的extrahaustorial膜包围(两者等人,2005年b; Bindschedler 等人,2016 )。

的....几项研究的乙菌˚FSP 大麦有这种真菌的特征在于基因表达图谱和蛋白质组(Bindschedler 等人,2011; Pennington et al。,2016)。然而, B. Graminis 的专性生物营养性质对这种真菌的研究提出了特殊的挑战,特别是对于haustoria的研究。吸器和外腔膜构成一个特殊的隔室,这对于与宿主的相互作用是功能上必不可少的(Wang et al。,2009; Pliego et al。,2013)。调查吸烟对于进一步了解营养摄入病原体的机制以及植物病原体识别至关重要。因此,从感染植物中获得纯化的吸收剂有可能对这些研究做出重大贡献。

以前,用于分离从采用亲和色谱上锈菌专biotrophs吸器的程序(Catanzariti 等人,2006; .. Garnica 等人,2014)。在90年代,引入了几种基于离心的方法从豌豆植物中分离出白粉病(Mackie et al。,1991; Testut et al。,1999)。这种方法应用后应用其他植物的白粉病,包括拟南芥(Wang et al。,2009; Micali et al。,2011)。最近,这些已发表方法的标志是它们与费力和相关的方法相关。戈弗雷

(2009)。此外,需要在冰上进行分离程序。在该方案中,我们开发了:隔离缓冲液,不同尺寸的钢网和多个离心步骤。更简单的方法以分离的 B.菌从感染的叶子大麦吸器。从植物细胞吸器释放通过酶促降解表皮细胞壁,和吸器通过过滤通过尼龙网纯化来实现。此方法需要消化宿主植物细胞壁,单次过滤和一次离心步骤。

关键字:白粉病, 大麦, 附生的, 表皮, 吸器提取, 纤维素酶, WGA-AF 488

材料和试剂

  1. 移液器吸头
  2. 专用封闭透明容器
  3. 剃刀刀片
  4. 封口膜
  5. 巴斯德吸管
  6. 15毫升/ 50毫升聚丙烯管
  7. 液氮容器
  8. 显微镜载玻片
  9. 盖玻片
  10. 大麦( Hordeum vulgare )cv.Golden Promise种子,大麦种子播种在John Innes 1堆肥中,混合蛭石(4:1)在12 x 12 cm盆中
  11. 液氮
  12. 醋酸纤维素
  13. 无水丙酮
  14. MES水合物粉末
  15. 盐酸
  16. WGA,Alexa Fluor 488缀合物(Thermo Fisher Scientific,目录号:W11261)
    注意:此染料显示Alexa Fluor ® 488染料的明亮绿色荧光(激发/发射最大值~495 / 519 nm)。
  17. Cellulase Onozuka R-10(Duchefa Biochemie,目录号:C8001)
    注意:来自绿色木霉的纤维素酶Onozuka R-10.1纤维素酶单位(U)将从羧甲基纤维素中释放1.0μmole葡萄糖。
  18. 氯化钠
  19. 氯化钾
  20. Na 2 HPO 4
  21. KH 2 PO 4
  22. PBS缓冲液(见配方1)

设备

  1. 镊子
  2. 移液器
  3. 剪刀
  4. 镊子
  5. 70微米尼龙网筛
  6. 恒温箱
  7. 离心分离机
  8. GFP过滤器
  9. 落射荧光显微镜
  10. -80°C冰柜

程序

  1. 解剖受感染的大麦叶
    1. 在20 oC的16小时光照/ 8小时黑暗周期中种植大麦( Hordeum vulgare )cv.Golden Promises种子。保持植物充分浇水至关重要,因为干旱胁迫可以防止有效解剖表皮。
    2. 在活的大麦植株上保存白粉病真菌( B. Graminis f。Sp。 hordei ),在一个专用的封闭透明容器中,保护气流免受可能取代分生孢子的气流的影响。通过在感染健康植物前一天摇动接种罐来分辨旧分生孢子。
    3. 在这个阶段,初生叶完全伸展。接种 B. Graminis 是通过在未感染的大麦上摇动重感染的植物进行的。植物将准备解剖接种后七天。
      注意:强烈建议在执行此程序时使用紧身面罩,以防止分生孢子进入操作员的呼吸道。
    4. 准备剪刀,镊子,剃刀刀片,Parafilm,巴斯德吸管,15毫升/ 50毫升聚丙烯管和液氮。将两个50毫升管放入液氮容器中,并加入液氮。
    5. 将5克醋酸纤维素溶于100毫升无水丙酮中,倒入15毫升聚丙烯管中。
    6. 用HCl调节pH至5.3。在室温下将20ml MES缓冲液倒入50ml管中,将0.2g MES水合物粉末溶于100ml H 2 O中,制成10mM MES缓冲液。
    7. 小心取出叶子并在取出叶子后等待几秒钟,等待几秒钟后取出叶子并等待几秒钟。然后将叶子放在两个巴斯德吸管上。
    8. 让丙酮从叶子表面蒸发5-8分钟。
      注意:此步骤至关重要:醋酸纤维素膜需要足够干燥才能去皮;但是,如果叶子太长而无法渲染,枯萎会导致表皮解剖不可能。
    9. 现在,将从叶子的其余部分除去附生的含有菌丝体的醋酸纤维素层的膜的剥离。在水上保持5分钟以确保充分保湿。
    10. 按照这个步骤,转动叶子,使正面朝上。在叶尖附近轻轻按下剃刀,小心地向前和向后摇动刀片。
      注意:这一步骤是破裂的,需要一些试验和错误才能获得足够的经验来实现正确的解剖。目的是切除上部,正面表皮和部分叶肉,同时保留下层
    11. 在抓住叶片尖端(这形成一个可用于拉动表皮的“突片”)并将切口保持在切口下方时,将背轴向上拉回。背面表皮,含有真菌吸器。
      注意:如果需要,可以通过用镊子推开其他层来释放表皮层。
    12. 一旦自由,小心地以连续平滑的方式剥离下表皮。
    13. 完成后,切开表皮并用镊子将其取下,取下叶尖(“标签”)。
      注意:要从表皮中分离吸器,请跳至步骤B1。
    14. 在液氮中的两个50ml聚丙烯管中分别将冻干的附生和硬膜外物质快速剥离以备将来使用。
    15. 储存在-80°C直至需要。

  2. Haustoria隔离
    1. 将漂浮的表皮置于50ml管中的15ml 10mM MES水合物缓冲液(pH5.3)中。
    2. 一旦收集到足够的表皮条(我们在视频1中使用一个盆),在新的50ml管中加入200mg纤维素酶R-10到10ml MES缓冲液中。充分混合,使纤维素酶的最终浓度为2%( / v)。
    3. 用镊子小心地将所有解剖的表皮转移到新的混合物中。
    4. 将悬浮液在28℃下孵育2小时,同时轻轻摇动(例如,在旋转平台上80rpm)。在孵育期间,纤维素酶将裂解上皮细胞并将吸收剂释放到孵育缓冲液中。
    5. 孵育后,将70微米尼龙网筛置于50毫升管上,缓慢地将缓冲液倒入过滤器中,同时使用镊子作为屏障,以保留较大块未消化的植物材料。
    6. 收集所有缓冲液后,在3,270 x g 的摇摆转子中离心10分钟,在4°C。
    7. 离心后,颗粒应含有吸器。
    8. 倒出上清液并保留颗粒状的吸器。
      注意:在此步骤之后,吸收剂可以在-80°C下长期储存或立即染色以显示(继续使用程序C)。

  3. WGA-Alexa Fluor 488染色(质量控制可选)
    1. 轻轻地将吸尘沉淀重悬于300μlPBS缓冲液(pH 7.4)中(配方1)。
    2. 将WGA-Alexa Fluor 488加入混合物中至终浓度为10μg/ ml。
    3. 通过倒置管几次充分混合。
    4. 将混合物装入显微镜载玻片上,小心地将盖玻片放在上面。
    5. 使用落射荧光显微镜在GFP过滤器下观察。


      视频1.将协议可视化为程序的视频记录

数据分析

代表性成果:上述方案提供了一种有效的酶促方法,可从宿主植物,大麦中获得 B. graminis 吸收剂。使用该方案获得的所有物质,包括附生菌丝体 - 含有醋酸盐条纹,有吸引力的流行病学果皮和纯化的吸附剂,可以在-80°C下储存,长期储存,直至需要进一步分析。一罐感染严重的大麦(大约80片叶子) 0.3克表皮和3-4×10 5 吸附剂(获得的吸烟剂的实际数量将取决于(NB包括醋酸纤维素薄膜,其构成该部分的主要部分,按重量计)通过WGA-Alexa Fluor染色后的落射荧光显微镜可以观察到分离的吸器的质量.488。大多数分离的吸器是高度支化的和完整的。足够小的植物碎片也可以通过70-μm尼龙网筛,因此在植物衍生的额外膜试验膜中第一次有限量的杂质,如图1B(箭头)所示。复杂的,例如叶绿体(图2,箭头)。


图1.用WGA-Alexa Fluor 488染色后可见的分离的吸器。 A.通过落射荧光显微镜观察并通过“GFP”过滤器观察到染色的吸器(荧光绿)。多个完整和成熟的吸器。 Bar =40μm.B。这显示了多重的吸器和围绕吸器的外围膜(箭头)。比例尺=10μm。


图2.带有一些杂质的分离的吸器过滤和离心后形成的颗粒不仅包括吸器,还包括一些植物碎片,例如叶绿体(自发荧光红,箭头)。比例尺=10μm。
叶绿体和细胞壁碎片。
过夜孵育产生最大数量的吸附剂:约5×10 (这被认为是thes)&nbsp;我们测试了纤维素酶总体消化时间的几种选择:1小时,2小时,3小时和过夜。孵化1小时后仅释放不到30%的过夜消化后获得的最大值。消化2小时和3次孵化最大量的吸附物我们可以实现),但也有更多的植物材料碎片降低了分离的吸器的纯度。赚取3-4 x 10 5 haustoria,占最大值的80%以上。

讨论:白粉病真菌具有挑战性,因为它们对宿主植物的生长和发育具有绝对的要求。尤其是对于宿主细胞内发育的饲养结构的吸收,尤其如此。 (Catanzariti et al。,2006; Micali et al。,2011)。以前已经发表了几种粉状霉菌吸附剂的亲和分离方法,包括 B. Graminis (Mackie et al。,1991; Micali et al。,2008)。然而,该方法证明了 B. graminis ,结合梯度离心和多次过滤,产生含80%吸器的颗粒;其余报告为细胞壁碎片和叶绿体(Godfrey 等人,,2009)。在以前的研究中,来自不同植物的大多数是通过酶的组合实现的,包括纤维素(Sun et al。,2013; Wu et al。,2017)。从大麦白粉病中获取相对纯净和完整的吸收剂,我们开发了一种快速有效的方法,依靠从叶子的其余部分解剖受感染的表皮,然后降解细胞壁和裂解表皮细胞。 &nbsp;
Barley cv.Golden promise因其对白粉病的高度接受而被选中以保持财务状况。适当数量的种子是值得的在这项研究中,我们使用大约3克种子用于10厘米直径的盆。为了确保高感染率,在幼苗密度的同时使用的植物不会太高而不能干扰初级叶的饱和接种。以这种方式,我们确保仅使用新鲜的分生孢子(<1天龄),导致终止地质学的高百分比成功,需要从用作接种物来源的植物中除去超过一天的分生孢子。年轻的叶子更容易解剖:在行人形成解剖的更高级阶段之间进行权衡取消对于孤立的成功是至关重要的。在实践中,我们发现在接种后7天(dpi)使用植物作为理想的折衷方案。此外,初生叶背面的表皮是我们经常只解剖原始叶片的背面表皮。与正面表皮或其他叶片的表皮相比,最容易解剖。 &nbsp;
增加纤维素酶浓度(高于2.5%)和各种酶(纤维素酶和macerozymes)的组合作为一组条件。及时iso iso iso iso iso iso iso iso iso iso iso iso iso iso iso按照惯例,我们使用2%的纤维素酶来分解细胞壁以释放吸器,然而,他们发现它们很容易导致分离结构的低生存力(Wu et al。,2017)。在比较不同的孵育时间后,我们在大多数情况下都有一个合适的折衷方案。临界培养的时间是从植物衍生结构中获得高分离的吸附和低污染。 &nbsp;
&nbsp;该协议将WGA-AF 488用于不锈钢分离的吸器,而不是使用考马斯蓝的传统方法。凝集素部分与可见的荧光结构和荧光结构荧光结构可见的真菌结构结合如果需要,可以通过用Calcofluor White染色来观察污染的植物细胞壁。 &nbsp;
&nbsp;我们认为该协议可适用于其他具有适当修改的白粉病。

食谱

  1. 1x PBS缓冲液
    8克NaCl
    0.2克KCl
    1.44g Na 2 HPO 4
    0.24克KH 2 PO 4
    溶于800毫升蒸馏水中 用HCl调节pH值至7.4 加入蒸馏水至总体积1L

致谢

LLC由中国奖学金委员会(CSC)的学生资助,BC由格雷戈里信托(伦敦帝国理工学院)资助。

竞争利益

作者声明,这里描述的工作没有相互竞争的利益。

参考

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Copyright: © 2019 The Authors; exclusive licensee Bio-protocol LLC.
引用:Li, L., Collier, B. and Spanu, P. D. (2019). Isolation of Powdery Mildew Haustoria from Infected Barley. Bio-protocol 9(14): e3299. DOI: 10.21769/BioProtoc.3299.
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