搜索

Wheat Coleoptile Inoculation by Fusarium graminearum for Large-scale Phenotypic Analysis
用于大规模表型分析的小麦胚芽鞘禾谷镰孢菌接种   

下载 PDF 引用 收藏 提问与回复 分享您的反馈 Cited by

本文章节

参见作者原研究论文

本实验方案简略版
The Plant Cell
Dec 2012

Abstract

The ascomycete fungus Fusarium graminearum is a destructive fungal pathogen of wheat, barley and maize. Although reverse genetics and homologous recombination gene deletion methods have generated thousands of gene deletion mutants of F. graminearum, evaluating virulence of these fungal mutants is still a rate-limiting step. Here we present a protocol for inoculation of wheat coleoptiles with conidial suspensions for large-scale phenotypic analysis, and describe how it can also be used to assess fungal infectious growth and symptom developmentat a cellular scale. The inoculation method described in this protocol provides highly reproducible results in wheat coleoptile infection by F. graminearum.

Keywords: Fusarium graminearum (禾谷镰孢菌), Wheat coleoptile (小麦胚芽鞘), Plant-fungal interaction (植物与真菌相互作用), Pathogenicity assays (致病性测定)

Background

Fusarium graminearum (previously also called Gibberella zeae) is a destructive pathogen that upon infection is responsible for causing Fusarium head blight (FHB) and seedling blight on cereal crops, as well as stalk and ear rot on maize (Dal Bello et al., 2002; Bai and Shaner, 2004; Kazan et al., 2012). Extensive molecular and genetic studies have been performed to investigate the interaction between F. graminearum and wheat. Given the availability of an efficient genetic transformation system and the well annotated genome, hundreds of F. graminearum genes have been investigated for their roles in vegetative growth, sexual development, secondary metabolism, stress responses and even virulence on host (Jia and Tang, 2015). However, only a few fungal effectors (e.g., FGL1 and deoxynivalenol) and host resistance genes (e.g., wheat Fhb1) have been identified (Proctor et al., 1995; Blümke et al., 2014; Rawat et al., 2016).

F. graminearum has been reported to lack pathogen-specialized patterns that typically induce gene-for-gene-mediated resistance in the host (van Eeuwijk et al., 1995). Furthermore, gene redundancy and functional complementation make assigning definitive virulence roles to pathogen genes achallenge. In addition, the traditional wheat head infection assay is limited due to seasonal, temporal and spatial factors. The distinct structures (rachis, paleas, lemmas, caryopses and glumes) and diverse features of wheat florets also make it difficult to track the infection progress of F. graminearum.

Previously, we used a modified wheat coleoptile infection assay and microscopic inspection to study F. graminearum infection inside host tissue (Zhang et al., 2012). Unlike the wheat head infection assay, there are few temporal and spatial constraints for the seedling infection system. The wheat coleoptile infection assay is performed in a growth chamber that can hold up to two hundred 24-well plates, which means more than 100 genes can be evaluated for their roles in virulence (three independent transgenic lines for each tested gene, and at least twelve seedlings for inoculation of each fungal strain). The time required to complete the assay is short: ten days for seed germination (Figure 1), inoculation (Figure 2) and examination of lesion size (Figure 3). The structure of wheat coleoptile is simple: the annular coleoptile comprises similar cells and two vascular bundles (Figure 2D) and is easy to inspect microscopically (Zhang et al., 2012). Seven genes were identified required for full virulence of F. graminearum on wheat coleoptile, and several of which were also required for wheat head infection (Zhang et al., 2012) and even maize stalk infection (Zhang et al., 2016).

Materials and Reagents

  1. Pipette tips (Corning, Axygen®, catalog number: T-300-R-S )
  2. Disposable paper towels (Vinda Classic Blue 1800, Vinda, catalog number: V4028 )
  3. 24-well cell culture plate (Corning, Costar®, catalog number: 3524 )
  4. Sterile toothpick
  5. Microtubes (Corning, Axygen®, catalog number: MCT-150-C )
  6. Medical-grade gauze (regular cotton yarn, Shanghai Honglong Medical Material Company)
  7. Medical-grade absorbent cotton (Shanghai Honglong Medical Material Company)
  8. Enamel tray (20x 30 x 5 cm)
  9. Glass slides (specifications: 76.2 x 25.4 mm, thickness: 1.0-1.2 mm) (Livingstone, catalog number: 7105-1 )
  10. Cover slips (specifications: 24 x 50 mm, thickness: 0.13-0.16 mm) (CITOTEST LABWARE MANUFACTURING, catalog number: 10212450C )
  11. Fungal strains: F. graminearum wild-type strain PH-1 (NRRL 31084), AmCyanPH-1 (Zhang et al., 2012) and gene deletion mutants of PH-1 (Zhang et al., 2012 and 2016)
  12. Plant material: Wheat (Triticum aestivum) cultivar Zhongyuan 98-68 (susceptible to F. graminearum and widely cultured in Henan, China)
  13. Sterile water
  14. V8 vegetable juice (CAMPBELL, catalog number: V8® ORIGINAL )
  15. Calcium carbonate (CaCO3)
  16. Agar powder
  17. Mung beans
  18. V8 juice agar medium (He et al., 2016; see Recipes)
  19. Mung bean liquid medium (He et al., 2016; see Recipes)

Equipment

  1. 500 ml flask
  2. Pipettes (Eppendorf)
  3. Growth chamber (Ningbo Jiangnan Instrument Factory, model: RXZ-1000 )
  4. Mould cultivation cabinet (Yiheng, model: MJ-150-I )
  5. Biological safety cabinet (ESCO Micro, model: FHC-1200A )
  6. Constant temperature shaker (Taicang, model: DHZ-DA )
  7. Hemocytometer (0.10 mm, 1/400 mm2) (QIUJING, model: XB-K-25 )
  8. Fluorescent microscope (Olympus, model: Olympus BX51 )
  9. Confocal microscope (Olympus, model: Fv10i )
  10. Centrifuge (Beckman Coulter, model: Avanti J-E Series )
  11. Camera (Canon, model: EOS 7D )
  12. Autoclave
  13. Rule

Software

  1. ImageJ (http://rsbweb.nih.gov/ij/index.html)
  2. Microsoft Excel

Procedure

  1. Preparation of wheat seedlings
    1. Soak the wheat seeds at room temperature in a 500 ml flask, and rinse with running water overnight (Figure 1A).
      Note: Prepare about 20 seeds for each inoculation treatment to make sure at least 12 germinated seedlings can be used for each inoculation treatment in one experiment. For example, to examine the virulence of two strains of mutant F. graminearum, along with one wild-type strain and mock inoculation, 80 seeds need to be soaked.
    2. Place the imbibed seeds in a medical enamel tray containing 2 layers of wet gauze (Figure 1B) and germinate in the dark for 1 day at 25 °C in a growth chamber.
      Note: Cover the tray with the lid to keep the gauze wet during germination.
    3. Put a small piece of paper towel in each well of 24-well cell culture plates and drench with water. Transplant the germinated seeds to 24-well cell culture plates, one seed per well (Figure 1C).
    4. Grow the plants in the growth chamber for 1 day under controlled environment conditions at 25 °C with a 12 h light/12 h dark photoperiod.


      Figure 1. Germination of wheat seeds. Wheat seeds were rinsed in flask with water (A) for one night and then transferred to an enamel tray with wet gauze (B). The germinated seeds were transplanted to 24-well cell culture plates (C). The ruler has scale in mm.

  2. Preparation of F. graminearum conidia suspension
    1. From a stock culture, pick a small quantity of F. graminearum mycelia using a sterile toothpick, and transfer to a fresh V8 agar plate. Incubate the culture in 25 °C growth chamber for 3-5 days.
    2. Scrape the V8 agar plate, now full of aerial hyphae with a sterilized tweezer, and pick up some pieces. Transfer these pieces into 100 ml sterilized mung bean liquid medium, then incubate in a constant temperature shaker at 25 °C and 150 rpm for 3-5 days.
    3. Filter the mung bean liquid medium culture, then collect the filtered liquid medium into a sterile 250 ml centrifuge bottle. Centrifuge at 7,500 x g for 10 min at room temperature.
    4. Discard the supernatant and re-suspend the conidia pellet in 1 ml sterile water by pipetting, then transfer the conidia suspension into a 1.5 ml sterile centrifuge tube. Wash the pellet three times using sterile water.
    5. Resuspend the conidia pellet in sterile water and adjust the concentration to 106 conidia/ml.
      Note: The suspension should be used for inoculation within 2 h. Resuspend the conidia just before inoculation.

  3. Wheat coleoptile inoculation
    1. Cut off the top 1-2 mm of coleoptiles of three-day-old wheat seedlings and add 2 μl F. graminearum wild-type or mutant suspension (106 conidia/ml) to the top of remaining seedling (Figure 2B and Video 1). For mock-inoculation controls, add 2 μl sterile water to the wounded sites.
      Note: Check the status of seedlings before inoculation. The coleoptiles of the seedlings ready for inoculation should have not been ruptured, see Figure 2A.


      Figure 2. Inoculation of wheat seedlings with conidia suspension. A. Wheat seedlings growing at day 3; B. Process for wheat seedling inoculation; C. Inoculated wheat seedlings; D. Cross section micrograph of wheat seedling. The image inserted in the middle of (B) shows microscopic examination of the conidia suspension on a hemocytometer just before inoculation. C: coleoptile; L: leaf; V: vascular bundle. Scale bars = 20 μm.

      Video 1. Process for wheat seedling inoculation

    2. Grow the inoculated plants in a growth chamber for another 7 days under controlled environment conditions at 25 °C with a 12 h light/12 h dark photoperiod and 95% relative humidity. The infection progress of fluorescently-tagged F. graminearum strains can be tracked under microscopy (step C4).
      Note: The lesions caused by F. graminearum are inconspicuous at the early infection stage (e.g., 1 day post inoculation, dpi). At 2-3 dpi, the coleoptiles at the inoculation sites are turning dark brown and there are aerial hyphae (Figure 3A).


      Figure 3. Symptom of F. graminearum infection on wheat seedlings. A. Infected wheat seedlings at 3 dpi (day post inoculation). White arrows show the lesions caused by F. graminearum. B. Growth of F. graminearum hyphae inside wheat coleoptiles. H: hyphae; PC: plant cell. White scale bars represent 20 μm. C. Infected wheat seedlings at 7 dpi; D. Lesion size caused by F. graminearum strains. Black scale bar represents 1 cm. Measurements of one representative experiment are shown. Data are means ± SD (n = 12), *P < 0.001 (significant), Student’s t-test.

    3. Measure the lesion size on coleoptiles of infected wheat seedlings at 7 dpi by photographing them with a ruler as reference (Figure 3C).
      Note: Repeat the experiment at least three times, each time use at least 12 seedlings per treatment.
    4. Microscopic observation of wheat coleoptile infection by fluorescently-tagged F. graminearum strains (optional).
      The conidia germinated on the coleoptile surface within 10 h-after-inoculation (HAI), and spread quickly inside host tissue from 16 HAI to 64 HAI (Zhang et al., 2012).
      1. Harvest wheat seedlings at given time point after inoculation, and cut off the coleoptiles according to the lesion size.
      2. Place coleoptiles on a glass slide immersed in sterile water covered by a coverslip.
      3. Examine coleoptiles using Olympus BX51 microscope or Olympus Fv10i as described in (Zhang et al., 2012; He et al., 2016).
    5. Chemical treatment (Figure 4) (optional).
      1. Prepare cotton strips (4 x 2 cm).
      2. Wrap the cotton strips around the top of the coleoptiles of 1 dpi wheat seedlings (Video 2).
      3. Add chemical solutions (amino acid solutions, etc.) to the cotton strips.
      4. Grow the wheat seedlings in a growth chamber for another 6 days under conditions described in step C2.


        Figure 4. Process for chemical treatment on wheat coleoptiles after inoculation. One day after inoculation, seedlings ready for chemical treatment are shown in the top panel. Note that new leaves have protruded from the coleoptile. Prepare cotton strips, and wrap the wound sites of the coleoptiles with cotton strips (middle panel). Then pipette chemical solutions (20-40 μl) to the wrapped cotton strips.

        Video 2. Wrapping the wheat seedling with cotton strip

Data analysis

The longitudinal length of brown lesions on wheat coleoptiles was measured as the lesion size (Figure 5) at the indicated time using ImageJ. Open the photo taken at 7dpi in ImageJ and choose straight line option to measure 1 cm of ruler in the photo as scale, and set the length as 1 cm. Then measure the length of brown lesions (Figure 5A) and export measurements to excel sheet (Figure 5B). Student’s t-test or one-way ANOVA was used to analyze the virulence with Microsoft Excel (Figure 5C). The diminished lesion size is interpreted as reduced virulence of F. graminearum on wheat coleoptiles (Figure 5D).


Figure 5. Lesion size measurements and statistical analysis. A. Representative image of seedlings after 7 days inoculation of wild-type (WT) or mutant (M1) strains of F. graminearum. Red lines indicate the ImageJ straight line tool measuring the lesion size. B. The sample data sets of lesion sizes of WT and M1 from three independent experiments in excel. C. Calculation of averages of experimental average lesion sizes and standard errors (SE), and perform t-test analysis. D. Results are charted in bar graph.

Notes

In step C2, it is important to maintain a high humidity environment during the infection progress of F. graminearum.

Recipes

  1. V8 juice agar medium (1L)
    168 ml V8 vegetable juice
    1 g CaCO3
    15 g agar powder
    Autoclave at 121 °C for 20 min
  2. Mung bean liquid medium (1 L)
    1. 40 g mung beans (Vignaradiata) (dried, available at grocery stores or supermarket)
    2. Put mung beans in boiling water, then boil for about 10 min and cool to room temperature
    3. Filter through gauze and discard the bean residue
    4. Add up to 1 L with distilled water
    5. Autoclave at 121 °C for 20 min

Acknowledgments

We thank Dr. Sheila McCormick for editing this protocol. This protocol was modified from previous inoculation method Wu et al., 2005 and Zhang et al., 2012. The research in the Tang lab was supported by the Ministry of Agriculture of China (Grant 2016ZX08009-003),the National Key Research and Development Program of China (Grant 2016YFD0100600), and the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB11020500).

References

  1. Bai, G. and Shaner, G. (2004). Management and resistance in wheat and barley to fusarium head blight. Annu Rev Phytopathol 42: 135-161.
  2. Blümke, A., Falter, C., Herrfurth, C., Sode, B., Bode, R., Schafer, W., Feussner, I. and Voigt, C. A. (2014). Secreted fungal effector lipase releases free fatty acids to inhibit innate immunity-related callose formation during wheat head infection. Plant Physiol 165(1): 346-358.
  3. Dal Bello, G., Mónaco, C. and Simón, M. (2002). Biological control of seedling blight of wheat caused by Fusarium graminearum with beneficial rhizosphere microorganisms. World J Microbiol Biotechnol 18(7): 627-636.
  4. He, J., Yuan, T.L. and Tang, W.H. (2016). Fusarium graminearum maize stalk infection assay and microscopic observation protocol. Bio Protoc 6(23): e2034.
  5. Jia, L. J. and Tang, W.H. (2015). The omics era of Fusariumgraminearum: opportunities and challenges. New Phytol 207:1-3
  6. Kazan, K., Gardiner, D. M. and Manners, J. M. (2012). On the trail of a cereal killer: recent advances in Fusarium graminearum pathogenomics and host resistance. Mol Plant Pathol 13(4): 399-413.
  7. Proctor, R. H., Hohn, T. M. and McCormick, S. P. (1995). Reduced virulence of Gibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene. Mol Plant Microbe Interact 8(4): 593-601.
  8. Rawat, N., Pumphrey, M. O., Liu, S., Zhang, X., Tiwari, V. K., Ando, K., Trick, H. N., Bockus, W. W., Akhunov, E., Anderson, J. A. and Gill, B. S. (2016). Wheat Fhb1 encodes a chimeric lectin with agglutinin domains and a pore-forming toxin-like domain conferring resistance to Fusarium head blight. Nat Genet 48(12): 1576-1580.
  9. van Eeuwijk, F. A., Mesterhazy, A., Kling, C. I., Ruckenbauer, P., Saur, L., Burstmayr, H., Lemmens, M., Keizer, L. C., Maurin, N. and Snijders, C. H. (1995). Assessing non-specificity of resistance in wheat to head blight caused by inoculation with European strains of Fusarium culmorum, F. graminearum and F. nivale using a multiplicative model for interaction. Theor Appl Genet 90(2): 221-228.
  10. Wu, A. B., Li, H. P., Zhao, C. S. and Liao, Y. C. (2005). Comparative pathogenicity of Fusarium graminearum isolates from China revealed by wheat coleoptile and floret inoculations. Mycopathologia 160(1): 75-83.
  11. Zhang, X. W., Jia, L. J., Zhang, Y., Jiang, G., Li, X., Zhang, D. and Tang, W. H. (2012). In planta stage-specific fungal gene profiling elucidates the molecular strategies of Fusarium graminearum growing inside wheat coleoptiles. Plant Cell 24(12): 5159-5176.
  12. Zhang, Y., He, J., Jia, L. J., Yuan, T. L., Zhang, D., Guo, Y., Wang, Y. and Tang, W. H. (2016). Cellular tracking and gene profiling of Fusarium graminearum during maize stalk rot disease development elucidates its strategies in confronting phosphorus limitation in the host apoplast. PLoS Pathog 12(3): e1005485.

简介

子囊菌真菌禾谷镰刀菌是小麦,大麦和玉米的破坏性真菌病原体。 尽管反向遗传学和同源重组基因缺失方法已经产生了成千上万的F基因缺失突变体。 评估这些真菌突变体的毒力仍然是一个速率限制的步骤。 在这里,我们提出了用于大规模表型分析用分生孢子悬浮液接种小麦胚芽鞘的方案,并且描述了如何也可以用它来评估细菌感染生长和症状发展。 本方案中描述的接种方法通过F提供了小麦胚芽鞘感染的高度可重复的结果。菌。
【背景】镰刀菌镰刀霉(以前也称为赤霉菌)是一种破坏性病原体,在感染后,导致镰孢霉病(FHB)和幼苗枯萎病(Dal Bello等人,2002; Bai和Shaner,2004; Kazan等人,2012年),谷物作物以及玉米的茎和耳腐。已经进行了广泛的分子和遗传学研究,以研究。F之间的相互作用。禾谷镰刀和小麦。鉴于有效的遗传转化系统和井注释基因组的可用性,数百种。已经研究了禾谷镰刀菌基因在营养生长,性发育,次生代谢,应激反应以及宿主中的毒力方面的作用(Jia and Tang,2015)。然而,已经鉴定了仅几种真菌效应物(例如,FGL1和脱氧雪腐镰刀菌烯醇)和宿主抗性基因(例如,小麦Fhb1 )(Proctor ,1995;Blümke等人,2014; Rawat等人,2016)。
F。据报道,禾谷镰刀菌缺乏通常在宿主中诱导基因为基因介导的抗性的病原体特异性模式(van Eeuwijk等人,1995)。此外,基因冗余和功能互补使病原体基因攻击具有明确的毒力作用。此外,传统的小麦头感染测定由于季节性,时间和空间因素的限制。独特的结构(rachis,oldas,lemmas,caryopses和glume)和小麦小花的多样特征也使得难以追踪F的感染进展。菌。
&NBSP;以前,我们使用改良的小麦胚芽鞘感染试验和显微镜检查来研究。疟原虫感染宿主组织内(Zhang等人,2012)。与小麦头感染测定不同,幼苗感染系统几乎没有时间和空间限制。小麦胚芽鞘感染测定在可以容纳多达两百个24孔板的生长室中进行,这意味着可以评估超过100个基因在毒力中的作用(每个测试基因的三个独立的转基因品系,至少用于接种每个真菌菌株的12个幼苗)。完成测定所需的时间很短:种子发芽10天(图1),接种(图2)和病变大小检查(图3)。小麦胚芽鞘的结构很简单:环状胚芽鞘包含相似的细胞和两个维管束(图2D),并且在显微镜下易于检查(Zhang等人,2012)。鉴定需要七种基因完全毒力的F基因。禾本科小麦胚芽鞘,其中几种也是小麦头部感染所必需的(Zhang等人,2012),甚至玉米秸秆感染(Zhang等人,,2016)。

关键字:禾谷镰孢菌, 小麦胚芽鞘, 植物与真菌相互作用, 致病性测定

材料和试剂

  1. 移液器吸头(Corning,Axygen ®,目录号:T-300-R-S)
  2. 一次性纸巾(Vinda Classic Blue 1800,Vinda,目录号:V4028)
  3. 24孔细胞培养板(Corning,Costar ®,目录号:3524)
  4. 无菌牙签
  5. 微管(Corning,Axygen ®,目录号:MCT-150-C)
  6. 医用纱布(普通棉纱,上海虹龙医疗材料公司)
  7. 医用级吸收棉(上海宏隆医疗材料公司)
  8. 搪瓷盘(20x 30 x 5厘米)
  9. 玻璃片(规格:76.2×25.4mm,厚度:1.0-1.2mm)(Livingstone,目录号:7105-1)
  10. 盖板(规格:24 x 50 mm,厚度:0.13-0.16 mm)(CITOTEST LABWARE MANUFACTURING,目录号:10212450C)
  11. 真菌菌株:F。葛根氏菌野生型菌株PH-1(NRRL 31084),AmCyanPH-1(Zhang等人,2012)和PH-1基因缺失突变体(Zhang等人。,2012和2016)
  12. 植物材料:小麦(小麦)品种中原98-68(易于禾谷镰刀菌,广泛培养于中国河南)
  13. 无菌水
  14. V8蔬菜汁(CAMPBELL,目录号:V8 ® ORIGINAL)
  15. 碳酸钙(CaCO 3 3)
  16. 琼脂粉末/ /
  17. 绿豆
  18. V8果汁琼脂培养基(He&amp; et al。,2016;见食谱)
  19. 绿豆液体培养基(He&et al。,,2016;见食谱)

设备

  1. 500毫升烧瓶
  2. 移液器(Eppendorf)
  3. 生长室(宁波江南仪器厂,型号:RXZ-1000)
  4. 模具栽培柜(益恒,型号:MJ-150-I)
  5. 生物安全柜(ESCO Micro,型号:FHC-1200A)
  6. 恒温摇床(太仓,型号:DHZ-DA)
  7. 血细胞计数器(0.10mm,1/400mm 2)(QIUJING,型号:XB-K-25)
  8. 荧光显微镜(Olympus,型号:Olympus BX51)
  9. 共焦显微镜(Olympus,型号:Fv10i)
  10. 离心机(Beckman Coulter,型号:Avanti J-E系列)
  11. 相机(佳能,型号:EOS 7D)
  12. 高压灭菌器
  13. 规则

软件

  1. ImageJ( http://rsbweb.nih.gov/ij/index。 html
  2. Microsoft Excel

程序

  1. 小麦苗的制备
    1. 将小麦种子在室温下浸泡在500ml烧瓶中,用流水冲洗过夜(图1A) 注意:为每次接种处理准备约20粒种子,以确保在一次实验中每次接种处理至少可以使用12个发芽幼苗。例如,为了检测两种突变型禾谷镰孢菌株的毒力,以及一种野生型菌株和模拟接种,需要浸泡80粒种子。
    2. 将吸入的种子放入包含2层湿纱布(图1B)的医用搪瓷盘中,并在黑暗中将孪生子在25℃下在生长室中放置1天。
      注意:用盖子盖住托盘,以防止发芽期间纱布潮湿。
    3. 将一小块纸巾放在24孔细胞培养板的每个孔中并用水浸泡。将发芽的种子移植到24孔细胞培养板中,每孔一个种子(图1C)
    4. 在25℃的受控环境条件下,用12小时光/ 12小时暗光周期,在生长室中种植植物1天。


      图1.小麦种子发芽。 将小麦种子在具有水(A)的烧瓶中漂洗一夜,然后转移到具有湿纱布(B)的搪瓷盘中。将发芽的种子移植到24孔细胞培养板(C)中。标尺的尺寸为mm。

  2. 分生孢子悬浮液的制备
    1. 从股票文化中,选择少量的 F。 graminearum 使用无菌牙签将菌丝体转移到新鲜的V8琼脂平板上。将培养物在25℃生长室中孵育3-5天
    2. 刮V8琼脂板,现在充满了无菌镊子的气生菌丝,并拿起一些碎片。将这些片转移到100ml灭菌的绿豆液体培养基中,然后在恒温振荡器中在25℃和150rpm下孵育3-5天。
    3. 过滤绿豆液体培养基,然后将过滤的液体培养基收集到无菌的250ml离心瓶中。在室温下以7,500×g离心10分钟。
    4. 弃去上清液,通过移液将分生孢子沉淀物重新悬浮在1 ml无菌水中,然后将分生孢子悬浮液转移到1.5 ml无菌离心管中。用无菌水清洗沉淀三次。
    5. 将分生孢子沉淀物重新悬浮于无菌水中,并将浓度调至10分钟/分钟/ ml。
      注意:悬浮液应在2小时内用于接种。在接种前重悬分生孢子。

  3. 小麦胚芽鞘接种
    1. 切断顶端1-2毫米的三日龄小麦幼苗的胚芽鞘,并加入2微升F。禾本科植物野生型或突变体悬浮液(10分钟/分生孢子/毫升)到剩余幼苗的顶部(图2B和视频1)。对于模拟接种对照,向受伤部位加入2μl无菌水 注意:接种前检查幼苗的状态。准备接种的幼苗的胚芽鞘应该没有破裂,见图2A。


      图2.小麦幼苗与分生孢子悬浮液的接种 A.在第3天生长的小麦幼苗; B.小麦接种过程C.接种小麦幼苗; D.小麦幼苗横截面显微照片。插入(B)中间的图像显示在接种之前血细胞计数器上的分生孢子悬浮液的显微镜检查。 C:coleoptile; L:叶V:血管束。比例尺= 20μm
      Video 1. Process for wheat seedling inoculation

      To play the video, you need to install a newer version of Adobe Flash Player.

      Get Adobe Flash Player


    2. 在受控环境条件下在25℃下,在12小时光/ 12小时暗光周期和95%相对湿度下,将生长室中的接种植物再培养7天。荧光标记的禾谷镰刀菌菌株的感染进展可以在显微镜下追踪(步骤C4)。
      注意:在早期感染阶段(例如,接种后1天,dpi),由禾谷镰刀菌引起的损伤是不明显的。在2-3dpi下,接种部位的胚芽鞘转为深棕色,有空气菌丝(图3A)。


      图3. 的症状 F。 小麦幼苗感染。白色箭头显示由 F引起的病变。菌。 B.增长。小麦胚芽鞘内的禾本科细菌菌丝菌丝。 H:菌丝PC:植物细胞。白色比例尺代表20μm。 C.感染小麦幼苗7dpi; D.由E.引起的病变大小。禾本科菌株。黑色比例尺表示1厘米。显示一个代表性实验的测量。数据是平均值±SD( = 12),* 0.001(重要),Student's -test。

    3. 通过用标尺作为参考,以7dpi测量受感染的小麦幼苗的胚芽鞘上的病变大小(图3C)。
      注意:重复实验至少三次,每次处理至少使用12个幼苗。
    4. 通过荧光标记的F显微镜观察小麦胚芽鞘感染。禾本科菌株菌株(可选)。
      分生孢子在10 h接种后(HAI)内在胚芽鞘表面发芽,并迅速在宿主组织内从16个HAI快速扩散至64个HAI(Zhang等人,2012)。
      1. 在接种后的给定时间收获小麦幼苗,并根据病变大小切断胚芽鞘。
      2. 将coleoptiles放在浸在被盖玻片覆盖的无菌水中的玻璃片上。
      3. 使用Olympus BX51显微镜或Olympus Fv10i检查胚芽鞘,如(Zhang等人,2012; He等人,2016)所述。
    5. 化学处理(图4)(可选)。
      1. 准备棉条(4 x 2厘米)。
      2. 将棉条包裹在1dpi小麦幼苗的胚芽鞘顶部(视频2)。
      3. 向棉条上加入化学溶液(氨基酸溶液,等)。
      4. 在步骤C2中描述的条件下,在生长室中种植小麦幼苗6天

        图4.接种后小麦胚芽鞘的化学处理方法。 接种后一天,准备进行化学处理的幼苗显示在上面板上。请注意,新叶子从胚芽鞘突出。准备棉条,并用棉条(中间板)包裹胚芽鞘的伤口部位。然后将化学溶液(20-40μl)移液到包裹的棉条上。

        Video 2. Wrapping the wheat seedling with cotton strip

        To play the video, you need to install a newer version of Adobe Flash Player.

        Get Adobe Flash Player

数据分析

使用ImageJ在指定时间测量小麦胚芽鞘上的褐色病变的纵向长度作为病变大小(图5)。打开在ImageJ中的7dpi拍摄的照片,并选择直线选项,以照片的尺寸测量1厘米的尺子,并将长度设置为1厘米。然后测量棕色病变的长度(图5A)并将测量值导出到Excel表(图5B)。使用Student's t 测试或单因素方差分析用Microsoft Excel分析毒力(图5C)。减小的病变大小被解释为F的毒力降低。禾谷镰刀菌在小麦胚芽鞘上(图5D)

图5.损伤尺寸测量和统计分析。 :一种。在野生型(WT)或突变体(M1)菌株接种7天后的幼苗的代表性图像。菌。红线表示ImageJ直线工具测量病变大小。 B.来自三个独立实验的WT和M1的病变大小的样本数据集。 C.计算实验平均病变大小和标准误差(SE)的平均值,并执行测试分析。 D.结果以条形图表示。

笔记

在步骤C2中,在F的感染进展期间保持高湿度环境是重要的。菌。

食谱

  1. V8果汁琼脂培养基(1L)
    168毫升V8蔬菜汁
    1克CaCO 3
    15克琼脂粉末 在121℃高压灭菌20分钟
  2. 绿豆液体培养基(1升)
    1. 40克绿豆(Vignaradiata )(干,可在杂货店或超级市场上购买)
    2. 将绿豆放入沸水中,然后煮沸约10分钟,冷却至室温
    3. 过滤纱布并丢弃豆渣
    4. 用蒸馏水加入1升
    5. 在121℃高压灭菌20分钟

致谢

我们感谢Sheila McCormick博士编辑这个协议。该方案从以前的接种方法Wu等人,2005年和Zhang等人于2012年进行了修改。唐实验室的研究得到农业部的支持中国(授权2016ZX08009-003),中国国家重点研究发展计划(授权2016YFD0100600)和中国科学院战略重点研究计划(XDB11020500)。

参考

  1. Bai,G。和Shaner,G。(2004)。小麦和大麦的管理和抵抗对镰刀菌枯萎病。 Annu Rev Phytopathol 42:135-161。
  2. Blümke,A.,Falter,C.,Herrfurth,C.,Sode,B.,Bode,R.,Schafer,W.,Feussner,I. and Voigt,CA(2014)。&lt; a class = -insertfile“href =”http://www.ncbi.nlm.nih.gov/pubmed/24686113“target =”_ blank“>分泌真菌效应物脂肪酶释放游离脂肪酸以抑制小麦头部感染期间先天免疫相关的胼lose质形成。植物生理学 165(1):346-358。
  3. Dal Bello,G.,Mónaco,C. andSimón,M。(2002)。&nbsp; 由具有有益根际微生物的禾本科镰刀菌引起的小麦幼苗枯萎的生物防治。世界J微生物生物技术 18(7): 627-636。
  4. 他,J.,Yuan,T.L。和唐,W.H. (2016)。 镰刀菌镰刀菌玉米秸秆感染试验和显微镜观察方案。 Bio Protoc 6(23):e2034。
  5. Jia,L.J.and Tang,W.H。 (2015)。镰刀菌的omics时代:机遇与挑战 207:1-3
  6. Kazan,K.,Gardiner,DM and Manners,JM(2012)。&nbsp; 在谷物杀手的踪迹上:镰刀菌镰孢病原体和宿主抗性的最新进展 Mol Plant Pathol 13(4):399- 413.
  7. Proctor,RH,Hohn,TM和McCormick,SP(1995)。&lt; a class =“ke-insertfile”href =“http://www.ncbi.nlm.nih.gov/pubmed/8589414”target =“ _blank“>由头孢霉烯毒素生物合成基因的破坏引起的玉米赤霉病的毒力降低。分子植物微生物相互作用 8(4):593-601。 br />
  8. Rawat,N.,Pumphrey,MO,Liu,S.,Zhang,X.,Tiwari,VK,Ando,K.,Trick,HN,Bockus,WW,Akhunov,E.,Anderson,JA and Gill,BS(2016 )。小麦Fhb1 编码具有凝集素结构域的嵌合凝集素和赋予对镰刀霉头病的抵抗力的造孔毒素样结构域。 Nat Genet 48(12):1576-1580 。
  9. van Eeuwijk,FA,Mesterhazy,A.,Kling,CI,Ruckenbauer,P.,Saur,L.,Burstmayr,H.,Lemmens,M.,Keizer,LC,Maurin,N。和Snijders,CH(1995)。 &nbsp; 评估小麦抗性的非特异性导致病害通过接种欧洲毒株镰刀菌,F。 graminearum 和 F。 nivale 使用乘法模型进行交互。 Theor Appl Genet 90(2):221-228。
  10. Wu,AB,Li,HP,Zhao,CS and Liao,YC(2005)。&nbsp; 小麦胚芽鞘和小花接种揭示的中国禾谷镰孢分离株的比较致病力。霉菌病160(1):75-83 。
  11. Zhang,XW,Jia,LJ,Zhang,Y.,Jiang,G.,Li,X.,Zhang,D. and Tang,WH(2012)。&nbsp; 在植物阶段特异性真菌基因谱中,阐明了小麦胚芽鞘内生长的禾谷镰刀菌的分子策略。 植物细胞 24(12):5159-5176。
  12. Zhang,Y.,He,J.,Jia,LJ,Yuan,TL,Zhang,D.,Guo,Y.,Wang,Y.and Tang,WH(2016)。&nbsp; 在玉米茎腐病发展过程中,禾谷镰刀菌的细胞跟踪和基因谱分析 其对抗宿主质粒中磷限制的策略。 PLoS Pathog 12(3):e1005485。
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Jia, L., Wang, W. and Tang, W. (2017). Wheat Coleoptile Inoculation by Fusarium graminearum for Large-scale Phenotypic Analysis. Bio-protocol 7(15): e2439. DOI: 10.21769/BioProtoc.2439.
提问与回复

(提问前,请先登录)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要谷歌账户来上传视频。

当遇到任务问题时,强烈推荐您提交相关数据(如截屏或视频)。由于Bio-protocol使用Youtube存储、播放视频,如需上传视频,您可能需要一个谷歌账号。