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Mar 2014

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Infection Process Observation of Magnaporthe oryzae on Barley Leaves
大麦叶片上稻瘟病菌感染过程观察   

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Abstract

Rice blast and wheat blast caused by Magnaporthe oryzae is a serious threat to rice and wheat production. Appropriate methods for observing M. oryzae infection process are important for study of the fungal infection mechanisms, plant resistance reactions, and host-M. oryzae interactions. The rice leaf sheath is commonly used to inoculate M. oryzae for observing the infection process, however, this method is a time-consuming and high technical work. Here, we describe an easier solution to observe M. oryzae infection process on barley leaves.

Keywords: Magnaporthe oryzae (稻瘟病菌), Rice blast fungus (稻瘟病菌), Barley leaf epidermis (大麦叶表皮), Inoculation (接种), Infection process (感染过程), Plant-pathogen interactions (植物-病原体相互作用)

Background

The filamentous fungus Magnaporthe oryzae can cause destructive rice blast and wheat blast diseases, which can also infect barley (Kohli et al., 2011; Dean et al., 2012). M. oryzae has been studied as a model to understand fungal-plant interactions (Yan and Talbot et al., 2016). This fungus initiates its infection by attaching the conidium to host surface, then the conidium germinates and forms a dome-shaped appressorium, by which it can penetrate into host cell for colonization (Wilson and Talbot, 2009). In host cells, the fungus colonizes as a biotrophic manner by forming bulbous and branched infection hyphae to interact with host defense system (Kankanala et al., 2007). M. oryzae sequentially invade living host cells and finally transformed into necrotrophic growth, during which the lesions appear and sporulation occurs. In order to study the fungal infection mechanism, or protein functions during infection, or plant defense reaction, it is required to observe cellular infection process of different strains in host cells. A rice leaf sheath method has been commonly used to observe the infection process (Koga et al., 2004), however, this method needs to waste a long time to prepare the rice leaf sheath, and the inoculation and sample preparation need a great deal of experience. Because barley is also the host of M. oryzae, and its leaf epidermis is easy for tearing, so we found that barley leaf epidermis method is an effective and simple method to observe the infection process of M. oryzae.

Materials and Reagents

  1. Petri dishes (6 cm and 9 cm diameters, ASONE)
  2. Lens paper (Fisher Scientific)
  3. Filter paper (Whatman)
  4. Medical gauze (Angyang Medical)
  5. Absorbent paper (Kimberly-Clark)
  6. 1.5 ml tubes (Eppendorf)
  7. Tips (10 μl, 200 μl and 1,000 μl, Axygen)
  8. Blades (Dexter Russell Cutlery, catalog number: 73-C )
  9. Glass slides (Fisher Scientific, catalog number: 12-550-343 )
  10. Coverslips (Fisher Scientific, catalog number: 12-547 )
  11. Pots (10 cm in diameter and 15 cm in height)
  12. Cultivated land soil
  13. Magnaporthe oryzae strains
    Note: The M. oryzae strains are maintained on dried filter paper pieces stock in -80 °C for long-term storage.
  14. Barley seeds (Hordeum vulgare, cv E9)
  15. Sterilized water (Milli-Q)
  16. Boiled oatmeal filtrate
  17. Tomato juice
  18. Agar (Sigma-Aldrich, catalog number: 17209 )
  19. Tween 20 (Sigma-Aldrich, catalog number: P9416 )
  20. Oatmeal tomato agar (OTA) solid media (see Recipes)
  21. 0.025% Tween 20 (see Recipes)

Equipment

  1. Scissor, tweezers, spreader
  2. Pipettes (Eppendorf, catalog numbers: 3124000016 [0.5-10 µl], 3124000032 [20-200 µl], and 3124000075 [100-1,000 µl])
  3. Incubation chamber (28 °C) for fungal growth and barley germination (Biolab Scientific, model: BIFG-101 )
  4. Hemocytometer (Marienfeld, catalog number: 0650030 )
  5. Greenhouse capable of temperature and humidity control for growing barley
  6. Optical microscope (Olympus, model: CX23 )
  7. Fluorescence microscope (Leica Microsystems, model: Leica DM2500 )
  8. Juice extractor
  9. Autoclave

Procedure

  1. Preparation of M. oryzae (Figure 1)


    Figure 1. Preparation procedure for conidia suspension of M. oryzae

    1. Inoculate the M. oryzae strains into solid OTA (Recipe 1) plates (6 cm diameter) and incubate for one week in an illumination incubator at 28 °C.
    2. Add 2 ml sterile distilled water containing 0.025% Tween 20 (Recipe 2) to each OTA plate and scrape with a spreader to harvest conidia. Use two layers of sterile lens paper to filter and remove hyphal fragments. Upon filtering, brief vortex by pipetting to re-suspend conidia.
      Note: Tween 20 will help to increase water hydrophobic property, therefore promote the formation of the water drops without collapse.
    3. Determine the conidia concentration by using a hemocytometer under an optical microscope, and adjust the final concentration to 1 x 105 per ml of 0.025% Tween 20 sterile water.

  2. Preparation of barley leaves
    1. Put barley seeds (around 50 seeds) into a Petri dish with an appropriate volume of sterile water and place the Petri dish in an incubation chamber at 28 °C for 12 h.
    2. Remove the water, and wash the seeds for several times with sterile water, then hung up in one layer gauze to dry for 6 h.
    3. Put the seed into the Petri dish (9 cm diameter), and immerse the seeds with an appropriate volume of sterile water, then cover several layers of wet gauze. Place the Petri dish in an incubation chamber for 12-18 h at 28 °C.
    4. After the seeds germinate, transfer them into pots (10 cm in diameter and 15 cm in height) containing 80% volume of cultivated land soil (20-30 seeds per pot). Place the pots in a greenhouse for growth at room temperature. Water the pot every two days to keep the soil wet. After one week, the barley leaves can be used for inoculation (Figure 2).


      Figure 2. Procedure for inoculation of M. oryzae on barley leaves

  3. Inoculation of barley with M. oryzae (Figure 2)
    1. Cut the barley leaves from the base with a scissor, then put them (back side up) in a Petri dish (9 cm diameter), which have been covered with two layers of water immersed absorbent paper. Fix the leaf base and tip with water immersed absorbent paper, and keep leaf flat (Video 1).
      Note: When fix the leaves base and tip with water immersed absorbent paper, keep the leaves dry and don’t touch the leaves with fingers or any other materials.

      Video 1. Put barley leaves into the Petri dish and fix with water immersed absorbent paper

    2. Drop the conidia suspension onto barley leaf beside the vein using a pipette (0.5-10 μl range) (Video 2).
      Note: Keep each drop with a diameter of around 2-3 μm and without collapse, if the drop is too large or collapsed, it may tend to the formation of aerial mycelium, but not appressorium. Keep the tips away from the epidermis could avoid collapse. Re-suspending conidia by shaking up and down of the tubes every time before absorbing the conidia suspension.

      Video 2. Drop conidia suspension onto the barley leaves

    3. Put on the lid, cover the Petri dishes with wetted absorbent papers, and place them into a sealed moist box. Then put the box in a dark incubator at 28 °C.

  4. Infection process observation (Figure 3)
    1. Takeout the barley leaves with a tweezer at different time points after inoculation. Use a blade to cross cut the leaf from up to down, at the tip side or base side. Keep the lower epidermis still connected, and subsequently tear down the lower epidermis (Figure 3A, Video 3).
      Note: During this process, try best not to touch the water drops, which could break the infection structures. Recommend observing penetration pegs and primary infection hyphae at 18 hpi, and secondary infection hyphae at 24-30 hpi.

      Video 3. Tear down the barley lower epidermis and put onto a glass slide

    2. Put the lower epidermis onto a glass slide, add some water to immerse the epidermis, then cover it with a coverslip.
    3. Observe the infection processes of M. oryzae in the cells of epidermis under a microscope, take images and calculate formation ratios of different infection structures (Figure 3A). For each time, more than 50 conidia should be calculated.
    4. Observe the subcellular localization of GFP-tagged proteins in infection structures under a fluorescence microscope (Figure 3B).
      Note: During observing, remember to add water when the sample is dried.


      Figure 3. Procedure for observing of different samples. A. Procedure of observing the infection process. B. Observation of protein subcellular localization. DIC, differential interference contrast; GFP, green fluorescent protein. Scale bars = 20 μm.

Data analysis

All information about data processing, statistical tests, replicates and independent experiments were already included in the original research paper (Chen et al., [2014], N-glycosylation of effector proteins by an α-1,3-mannosyltransferase is required for the rice blast fungus to evade host innate immunity. Plant Cell 26(3):1360-1376. doi: 10.1105/tpc.114.123588).

Notes

  1. Plant healthy is very important for the success of the infection assay. Barley plant should grow under adequate sunshine, and avoid soil drought. Otherwise, the barley epidermis can’t be well infected with M. oryzae.
  2. Accurately addition of 0.025% Tween 20 into the sterile water is important for successful inoculation. The tips should be cut off before absorbing the reagent. Absorb the Tween 20 slowly and the adhering reagent on tips should be removed.
  3. To prevent the drops from collapse during inoculation steps, the Petri dish should be moved as slowly as possible.

Recipes

  1. Oatmeal Tomato Agar (OTA) solid media (1 L)
    40 g boiled oatmeal filtrate
    150 ml tomato juice
    20 g agar
    1. Boil the oatmeal in 800 ml distilled water for 30 min, then filtrate the mixture by double-layer gauze
    2. Extract tomato juice by an extractor, then also filtrate the mixture by double gauze. Mix 150 ml tomato juices with boiled oatmeal filtrate, then add water to 1 L
    3. Add 20 g agar, and autoclave for 40 min
  2. 0.025% Tween 20
    Add 250 μl Tween 20 in 800 ml sterilized distilled water and adjust the volume to 1 L

Acknowledgments

The protocol was adapted from Chen et al. (2014). This work was supported by grants from the National Natural Science Foundation of China (31571952, 31601585). The author declares no conflict of interests.

References

  1. Chen, X. L., Shi, T., Yang, J., Shi, W., Gao, X., Chen, D., Xu, X., Xu, J. R., Talbot, N. J. and Peng, Y. L. (2014). N-glycosylation of effector proteins by an alpha-1,3-mannosyltransferase is required for the rice blast fungus to evade host innate immunity. Plant Cell 26(3): 1360-1376.
  2. Dean, R., Van Kan, J. A., Pretorius, Z. A., Hammond-Kosack, K. E., Di Pietro, A., Spanu, P. D., Rudd, J. J., Dickman, M., Kahmann, R., Ellis, J. and Foster, G. D. (2012). The Top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol 13(4): 414-430.
  3. Kankanala, P., Czymmek, K. and Valent, B. (2007). Roles for rice membrane dynamics and plasmodesmata during biotrophic invasion by the blast fungus. Plant Cell 19(2): 706-724.
  4. Koga, H., Dohi, K., Nakayachia, O. and Moria, M (2004). A novel inoculation method of Magnaporthe grisea for cytological observation of the infection process using intact leaf sheaths of rice plants. Physiol Mol Plant Pathol 64: 67-72.
  5. Kohli, M. M, Mehta, Y. R., Guzman, E, De Viedma, L. and Cubilla, L. E. (2011). Pyricularia blast – a threat to wheat cultivation. Czech J Genet Plant Breed 47:130-134.
  6. Wilson, R. A. and Talbot, N. J. (2009). Under pressure: investigating the biology of plant infection by Magnaporthe oryzae. Nat Rev Microbiol 7(3): 185-195.
  7. Yan, X. and Talbot, N. J. (2016). Investigating the cell biology of plant infection by the rice blast fungus Magnaporthe oryzae. Curr Opin Microbiol 34: 147-153.

简介

由稻瘟病引起的稻瘟病和小麦瘟病对水稻和小麦生产构成严重威胁。 观察 M的适当方法。 oryzae感染过程对于研究真菌感染机制,植物抗性反应和宿主M很重要。 oryzae 相互作用。 水稻叶鞘通常用于接种M。 oryzae 用于观察感染过程,然而,这种方法是一项耗时且高技术性的工作。 在这里,我们描述一个更容易观察 M的解决方案。 oryzae 在大麦叶子上的感染过程。

【背景】丝状真菌Magnaporthe oryzae可引起破坏性稻瘟病和小麦瘟病,其也可感染大麦(Kohli等人,2011; Dean等人, / em>,2012)。 微米。 oryzae已被研究作为了解真菌与植物相互作用的模型(Yan和Talbot等人,2016年)。这种真菌通过将分生孢子附着到宿主表面来启动其感染,然后分生孢子萌发并形成圆顶状附着胞,从而其可以穿入宿主细胞进行定植(Wilson和Talbot,2009)。在宿主细胞中,真菌通过形成球根和分枝的感染菌丝以与宿主防御系统相互作用而作为生物营养方式定殖(Kankanala等人,2007)。 微米。 oryzae 依次侵入宿主细胞并最终转化为坏死性生长,在此期间病变出现并发生孢子形成。为了研究感染过程中的真菌感染机制或蛋白质功能或植物防御反应,需要观察宿主细胞中不同菌株的细胞感染过程。水稻叶鞘法已被普遍用于观察感染过程(Koga等人,2004),然而,该方法需要浪费很长时间来制备水稻叶鞘,并且接种并且样品制备需要大量的经验。因为大麦也是M的主人。 oryzae ,其叶表皮容易撕裂,因此我们发现大麦叶表皮法是观察M感染过程的有效简单方法。米曲霉。

关键字:稻瘟病菌, 稻瘟病菌, 大麦叶表皮, 接种, 感染过程, 植物-病原体相互作用

材料和试剂

  1. 培养皿(6厘米和9厘米直径,ASONE)
  2. 镜头纸(Fisher Scientific)
  3. 滤纸(Whatman)
  4. 医用纱布(Angyang Medical)
  5. 吸水纸(Kimberly-Clark)
  6. 1.5毫升管(Eppendorf)
  7. 提示(10μl,200μl和1,000μl,Axygen)
  8. 刀片(Dexter Russell Cutlery,目录号:73-C)
  9. 玻璃载玻片(Fisher Scientific,目录号:12-550-343)
  10. Coverslips(Fisher Scientific,目录号:12-547)

  11. 壶(直径10厘米,高15厘米)
  12. 耕地土壤
  13. Magnaporthe oryzae 菌株
    注:米曲霉菌株在-80°C的干燥滤纸片上保存,以备长期保存。
  14. 大麦种子( Hordeum vulgare ,cv E9)
  15. 无菌水(Milli-Q)
  16. 煮燕麦滤液
  17. 番茄汁
  18. 琼脂(Sigma-Aldrich,目录号:17209)
  19. 吐温20(Sigma-Aldrich,目录号:P9416)
  20. 燕麦番茄琼脂(OTA)固体培养基(见食谱)
  21. 0.025%吐温20(见食谱)

设备

  1. 剪刀,镊子,吊具
  2. 移液器(Eppendorf,产品目录号:3124000016 [0.5-10μl],3124000032 [20-200μl]和3124000075 [100-1,000μl])
  3. 用于真菌生长和大麦萌发的培养箱(28℃)(Biolab Scientific,型号:BIFG-101)
  4. 血细胞计数器(Marienfeld,目录号:0650030)
  5. 温室能够控制温度和湿度,用于种植大麦
  6. 光学显微镜(奥林巴斯,型号:CX23)
  7. 荧光显微镜(徕卡显微系统,型号:Leica DM2500)
  8. 榨汁机
  9. 高压灭菌器

程序

  1. 准备 M。 oryzae (图1)


    图1.制备 M分生孢子的方法。 oryzae

    1. 接种 M。 oryzae 菌株转化为固体OTA(配方1)平板(直径6厘米),并在28°C的光照培养箱中培养一周。
    2. 加入含有0.025%吐温20(方案2)的2ml无菌蒸馏水至每个OTA平板并用撒布器刮取收获分生孢子。使用两层无菌镜头纸过滤和去除菌丝碎片。过滤后,通过移液短暂涡旋以重新悬浮分生孢子。
      注:吐温20将有助于增加水的疏水性,从而促进水滴的形成而不会塌陷。
    3. 在光学显微镜下使用血细胞计数器测定分生孢子浓度,并将终浓度调节至每毫升0.025%吐温20无菌水中1×10 5个/毫升。

  2. 大麦叶片的制备
    1. 将大麦种子(大约50粒种子)放入含有适量无菌水的培养皿中,并将培养皿置于28°C孵育室中12小时。
    2. 取出水,用无菌水洗几次种子,然后挂在一层纱布上干燥6小时。
    3. 将种子放入培养皿(9厘米直径),并用适量的无菌水浸种,然后覆盖几层湿纱布。
      将培养皿置于培养箱中28°C孵育12-18小时。
    4. 种子发芽后,将其转移到含有80%体积耕地土壤(每盆20-30粒种子)的盆中(直径10厘米,高15厘米)。将盆放在室温下生长的温室中。每两天浇水一次,以保持土壤湿润。一周后,大麦叶片可用于接种(图2)。


    图2.接种 M的程序。 oryzae 在大麦叶上

  3. 用大麦接种大麦。 oryzae (图2)
    1. 用剪刀从基座上切下大麦叶片,然后将它们(背面朝上)放入已用两层水浸吸水纸覆盖的培养皿(直径9厘米)中。
      使用水浸式吸水纸固定叶片底部和吸头,并保持叶片平整(视频1)。
      注意:使用水浸式吸水纸固定叶片基座和吸头时,请保持叶片干燥,并且不要用手指或任何其他材料接触叶片。



    2. 使用移液管(0.5-10μl范围)将分生孢子悬浮液滴在静脉旁的大麦叶上(视频2)。
      注意:如果滴剂过大或塌陷,保留每个滴剂的直径约2-3μm并且没有塌陷,这可能倾向于形成气生菌丝体,而不是附着体。保持提示远离表皮可以避免崩溃。每次吸收分生孢子悬浮液之前,通过摇动管子来重新悬浮分生孢子。



    3. 盖上盖子,用湿润的吸水纸覆盖培养皿,并将它们放入密封的潮湿箱中。然后将盒子置于28°C的黑暗培养箱中。

  4. 感染过程观察(图3)
    1. 接种后在不同时间点用镊子取出大麦叶片。使用刀片从顶部或底部横向切割叶片。保持下表皮连接,然后拆下下表皮(图3A,视频3)。
      注意:在这个过程中,尽量不要碰水滴,这可能会破坏感染结构。建议在18 hpi观察穿透钉和原发感染菌丝,并在24-30 hpi时观察继发感染菌丝。



    2. 将下表皮放在载玻片上,加入一些水浸泡表皮,然后盖上盖玻片。
    3. 观察 M的感染过程。 oryzae 在显微镜下的表皮细胞中,拍摄图像并计算不同感染结构的形成比例(图3A)。
      每次应计算超过50个分生孢子

    4. 在荧光显微镜下观察感染结构中GFP标记蛋白的亚细胞定位(图3B)。
      注意:在观察过程中,记得在样品干燥时加水。


      图3.观察不同样品的程序 A.观察感染过程的程序。 B.蛋白质亚细胞定位的观察。 DIC,差分干涉对比;绿色荧光蛋白,绿色荧光蛋白。比例尺= 20μm。

数据分析

关于数据处理,统计检验,重复和独立实验的所有信息已经包含在最初的研究论文中(Chen等人,[2014],通过α-1对效应蛋白进行N-糖基化,植物细胞“26(3):1360-1376。doi:10.1105 / tpc.114.123588),稻瘟病菌需要3-甘露糖基转移酶来避免宿主先天免疫。

笔记

  1. 植物健康对感染检测的成功非常重要。大麦植物应在充足的阳光下生长,并避免土壤干旱。否则,大麦表皮不能很好地感染M。 oryzae 。
  2. 准确添加0.025%吐温20到无菌水中对于成功接种是重要的。在吸收试剂之前应该切断吸头。缓慢吸收吐温20,并将尖端上的附着剂去除。
  3. 为了防止接种阶段液滴塌陷,培养皿应尽可能慢地移动。

食谱

  1. 燕麦番茄琼脂(OTA)固体培养基(1升)
    40克煮燕麦片过滤

    150毫升番茄汁 20克琼脂
    1. 将燕麦片在800毫升蒸馏水中煮30分钟,然后用双层纱布过滤混合物
    2. 用提取器提取番茄汁,然后用双纱布过滤混合物。将150毫升番茄汁和煮沸的燕麦片滤液混合,然后加水至1升
    3. 加入20克琼脂和高压灭菌器40分钟
  2. 0.025%吐温20

    在800毫升无菌蒸馏水中加入250μlTween 20并将体积调至1 L

致谢

该协议改编自Chen等人(2014年)。这项工作得到了国家自然科学基金(31571952,31601585)的资助。作者声明不存在利益冲突。

参考

  1. Chen,X.L.,Shi,T.,Yang,J.,Shi,W.,Gao,X.,Chen,D.,Xu,X.,Xu,J.R.,Talbot,N.J.和Peng,Y.L。(2014)。 稻瘟病需要通过α-1,3-甘露糖基转移酶对效应蛋白进行N-糖基化真菌来逃避宿主先天免疫。植物细胞 26(3):1360-1376。
  2. Dean,R.,Van Kan,JA,Pretorius,ZA,Hammond-Kosack,KE,Di Pietro,A.,Spanu,PD,Rudd,JJ,Dickman,M.,Kahmann,R.,Ellis,J。和Foster ,GD(2012)。 分子植物病理学中十大真菌病原体分子植物病理学< / em> 13(4):414-430。
  3. Kankanala,P.,Czymmek,K.和Valent,B。(2007)。 在真菌侵袭性侵袭过程中水稻膜动力学和胞间连丝的作用 植物细胞 19(2):706-724。
  4. Koga,H.,Dohi,K.,Nakayachia,O。和Moria,M(2004)。 用于细胞学观察感染过程的 Magnaporthe grisea 的新颖接种方法使用完整的水稻植物叶鞘。 Physiol Mol Plant Pathol 64:67-72。
  5. Kohli,M.M,Mehta,Y.R.,Guzman,E,De Viedma,L.和Cubilla,L.E。(2011)。 Pyricularia blast - 对小麦的种植构成威胁 捷克J Genet Plant品种 47:130-134。
  6. Wilson,R.A。和Talbot,N.J。(2009)。 承受压力:通过 Magnaporthe oryzae 调查植物感染的生物学。< / Rev> Nat Rev Microbiol 7(3):185-195。
  7. Yan,X.和Talbot,N.J。(2016)。 调查稻瘟病菌对植物感染的细胞生物学 Magnaporthe oryzae 。 Curr Opin Microbiol 34:147-153。
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引用:Chen, X. (2018). Infection Process Observation of Magnaporthe oryzae on Barley Leaves. Bio-protocol 8(9): e2833. DOI: 10.21769/BioProtoc.2833.
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