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Haustorium Induction Assay of the Parasitic Plant Phtheirospermum japonicum
寄生植物日本松蒿的吸器诱导测定

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Abstract

Phtheirospermum japonicum is a facultative root parasitic plant in the Orobanchaceae family used as a model parasitic plant. Facultative root parasites form an invasive organ called haustorium on the lateral parts of their roots. To functionally characterize parasitic abilities, quantification of haustorium numbers is required. However, this task is quite laborious and time consuming. Here we describe an efficient protocol to induce haustorium in vitro by haustorium-inducing chemicals and host root exudate treatments in P. japonicum.

Keywords: Parasitism(寄生), Parasitic plant(寄生植物), Orobanchaceae(列当科), Haustorium(吸器), Root(根), Host(宿主), Exudate(渗出物), DMBQ (DMBQ)

Background

Parasitic plants have evolved to obtain nutrients from other plants. Some of parasitic plants cause a significant damage to agriculture by infecting commercial crops (Spallek et al., 2013). Obligate parasitic plants require hosts to complete their lifecycle, while facultative parasitic plants can survive without hosts as autotrophic organisms but shift to heterotrophic by infection if host plants are nearby (Westwood et al., 2010). The common characteristic of all parasitic plants is a specialized organ called haustorium, which connects parasite with host by establishing vascular bridges (Saucet and Shirasu, 2016; Yoshida et al., 2016). Obligate root parasites form terminal haustoria that are derived from enlarged root tips, while facultative root parasites form lateral haustoria, which develop at the lateral side of the parasite roots without affecting the root meristem. Therefore, several lateral haustoria can form in a root. The early stage of haustorium development is characterized by enlarged root tissues caused by a combination of cell expansion and cell division. Several host-derived substances that are able to induce haustorium formation in vitro were previously identified. Such substances are called haustorium-inducing factors (HIF). Among them, the most active HIF is DMBQ (2,6-Dimethoxy-1,4-benzoquinone), initially isolated from sorghum root extracts (Chang and Lynn, 1986). Phtheirospermum japonicum, a facultative parasitic plant in the Orobanchaceae, is an ideal model to study the molecular mechanisms of the parasitism, because of its short life cycle, small size, and simple genetics as a selfing plant (Ishida et al., 2011; Cui et al., 2016). In addition, genetic manipulation of P. japonicum is now feasible (Ishida et al., 2011) and its large-scale transcriptome information is also available (Ishida et al., 2016). Here we report an efficient in vitro method for haustorium induction to investigate functionality of haustorium-related genes in P. japonicum. This method presents a step-by-step protocol for haustorium induction in vitro by DMBQ treatment or by contact with host exudates. This technique is useful to understand the genetic factors that trigger haustorium formation in parasitic plants.

Materials and Reagents

  1. Falcon 50 ml conical centrifuge tube (e.g., Corning, Falcon®, catalog number: 352070 )
  2. Filter paper, No. 2, Ø9 cm (Advantec, catalog number: 00021090 )
  3. Sterilized plastic plate dish with diameter of 100 mm (e.g., BioLite φ100 TC Dish, Fisher Scientific, catalog number: 12-556-002 )
    Manufacture: Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 130182 .
  4. Eppendorf® microcentrifuge tube (1.5 ml) (e.g., Fisher Scientific, catalog number: 05-408-129 )
  5. Surgical tape (or Parafilm) (e.g., 3M, catalog number: 1530-1 )
  6. Kitchen aluminium foil
  7. Sterilized square plastic plate dish 140 x 100 x 14.5 mm (Eiken Chemical, catalog number: AW2000 703077 )
  8. Microscope slides (e.g., Fisher Scientific, catalog number: 12-549-3 )
  9. Microscope coverslips L x W x D: 22 x 70 x 1.0 mm (e.g., Fisher Scientific, catalog number: 10-016-24 )
  10. Gloves
  11. Rice seeds (Oryza sativa japonica variety Nipponbare)
  12. Phtheirospermum japonicum seeds
  13. Commercial hypochlorite solution (Kao Japan) (approx. 6% sodium hypochloride)
  14. Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: 484016 )
  15. Tween 20 (Sigma-Aldrich, catalog number: P9416 )
  16. Water (Milli-Q grade)
  17. Propidium iodide (Sigma-Aldrich, catalog number: P4170 )
  18. Murashige and Skoog salts (Pre-mixed) (Wako Pure chemical Industries, catalog number: 392-00591 )
  19. Sucrose (Merck Millipore, catalog number: 107687 )
  20. Myo-inositol (Sigma-Aldrich, catalog number: I7508 )
  21. 2,6-dimethoxy-1,4-benzoquinone (DMBQ) (Sigma-Aldrich, catalog number: 428566 )
  22. Agar (Merck Millipore, catalog number: 101614 )
  23. Dimethyl sulfoxide (DMSO) (Wako Pure chemical Industries, catalog number: 041-29351 )
  24. Chloral hydrate (Sigma-Aldrich, catalog number: V000554 )
  25. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  26. GM media (see Recipes)
  27. DMBQ stock solution (10 mM) (see Recipes)
  28. Chloral hydrate solution (see Recipes)

Equipment

  1. Rice husker (Fujiwara Scientific, model: Testing rice husker )
  2. Tube rotator (e.g., TITEC, model: RT-50 , catalog number: 0000165-000)
  3. Laminar flow hood (e.g., YAMATO SCIENTIFIC, model: CCV-1300E )
  4. Plant growth chamber (e.g., NKsystem, model: LPH-411SP )
  5. Daylight-white fluorescent lamp (NEC LIGHTING, model: FL40SEX-N-HG )
  6. Microscope (Leica Microsystems, model: TCS-SP5 II )
  7. Vortex shaker (e.g., Scientific Industries, model: Vortex-Genie2 , catalog number: G560-SI-0246 2)
  8. Surgical scalpel handle (e.g., Swann-Morton, catalog number: 0933 )
  9. Surgical scalpel blade number 11 (e.g., Swann-Morton, catalog number: 0303 )
  10. Stainless steel forceps (e.g., Sigma-Aldrich, catalog number: F4142-1EA )
  11. Semi-analytical balance (e.g., Shimadzu, model: AUW220D )
  12. Water bath (e.g., Fisher Scientific, model: Fisher ScientificTM IsotempTM General Purpose Deluxe Water Bath, catalog number: S28124 )
  13. Light stereo microscope (e.g., Carl Zeiss, model: Stemi-2000 )
  14. Light microscope (e.g., Olympus, model: BX53-P )
  15. Autoclave (e.g., Hirayama, model: HG series )

Procedure

  1. Rice seed germination
    1. Remove the seed coats from rice seeds by a rice husker.
      Note: If the rice husker is not available, it is possible to remove the seed coat one by one by carefully pinching the seed’s awn with forceps or hands and pushing it down.
    2. Place the coat-removed seeds in a 50 ml plastic tube.
      Note: Maximum of ~20-30 seeds per tube.
    3. To surface sterilize the rice seeds, immerse them in 50% (v/v) commercial hypochlorite solution (~3% sodium hypochlorite in final solution) with 0.1% (v/v) Tween 20. Shake the seeds using a vortex set at maximum speed for 5 min and place the plastic tube in a tube rotator for 25 min set to 10-20 rpm.
    4. From this step, the procedures should be done in a sterile laminar flow hood, wash the seeds with sterilized deionized water (Milli-Q grade) for five times.
    5. Transfer the surface-sterilized seeds on a sheet of moistened filter paper placed in a plastic Petri dish (Ø100 mm). For 50 seeds use 12-15 ml of sterilized water to moisten the filter paper.
    6. Place the seeds in a growth chamber set at 26 °C under 16 h light and 8 h dark condition for 1 week with a light intensity of approximately 701 mol/m2/sec illuminated by the daylight-white fluorescence lamp (FL40SEX-N-FL, NEC, Japan).


      Figure 1. Rice root exudate treatment. A. One-week-old rice roots excised by scalpel were immobilized into agar block. The agar block was cut into small blocks (~2 x ~4 cm), each one contained around 20-25 mg of excised rice root. It was placed on P. japonicum plants maintained in vertical position for 2 weeks. B. Enlarged picture of (A). C. P. japonicum root without haustorium induction treatment; D and E. Induced haustoria (pointed by white arrows) formed along P. japonicum roots; F. Root tissues were cleared with chloral hydrate solution and observed under a microscope. G and H. The haustorium formed in a transgenic P. japonicum roots harbouring the construct ProPjYUC3:3xVENUS-N7-Pro35S:RFP, the merged filter(G) and YFP filter (H) photographs were taken under confocal Leica TCS-SP5 II microscopy. For details, see the reference Ishida et al. (2016). The white bars represent 1 cm and black bars 2.5 mm.

  2. P. japonicum seed germination
    1. Place the seeds in a 1.5 ml plastic tube (maximum of ~100 seeds per tube).
    2. Sterilize the surface of seeds by immersing in 10% (v/v) commercial hypochlorite solution (~0.6% sodium hypochlorite in final solution). Place the tube in a vortex shaker set at maximum speed for 1 min. Replace the solution with new bleach solution and keep in a vortex shaker for 9 min.
    3. From this step, the procedures should be done in a sterile laminar flow hood, wash the seeds with sterile-deionized water for five times.
    4. Immerse the seeds in sterile-deionized water. Keep them in darkness at 4 °C for overnight.
    5. Use the 1 ml tip to sow seed one by one on the top of a square plate dish containing GM media, around 12-15 seeds per plate. Seal the square plates with a surgical tape and cover it with aluminium foil. Keep the seeds in darkness for three more days to stimulate germination.
    6. Remove partially the aluminium foil, keeping the root parts covered.
    7. Place the square dish vertically in a growth chamber set at 25 °C under 16 h light and 8 h dark condition for 2 weeks.
      Note: It is important to let the parasitic plant to grow vertically to avoid the root to penetrate the agar media.
  3. Haustorium induced by rice root exudate
    1. Excise the roots from 1-week-old rice seedlings, using sterilized forceps and scalpel on sterilized plate dish (Ø9 cm).
    2. Measure the root fresh weight. For 180 mg of fresh weight root tissues, 20-25 excised roots are necessary.
    3. In a sterilized plate dish (Ø9 cm), 180 mg of fresh weight rice roots and 5 ml water from the plate where rice seeds were germinated were placed.
    4. Using sterilized scalpel, cut the roots into small pieces (around ~1-3 mm of length).
    5. Add 20 ml of 0.8% (w/v) agar, warmed in a water bath set at 60 °C.
    6. Mix well until the root pieces are evenly distributed across the agar.
    7. Let the agar to solidify for 30 min.
    8. Cut the agar block to approximately ~2 x ~4 cm and place them on the parasitic plant roots growing on GM media. The size of agar block should be enough to cover the parasitic roots. (Figures 1A and 1B)
      Note: When using transgenic roots, they are transferred to a plate dish (Ø9 cm) filled with 30 ml of 0.8% (w/v) agar with antibiotic cefotaxime (300 µg/ml). The agar block containing chopped rice roots and exudates should be put on the transgenic roots.
    9. The haustorium will be formed within 24 h (Figures 1D to 1F). Examples of transgenic roots forming haustoria are shown (Figures 1G to 1H).
    10. Remove the agar containing rice roots and count the haustorium number under a stereomicroscope.
  4. Haustorium induced by DMBQ
    1. Dilute the 10 mM DMBQ stock solution to 10 µM DMBQ working solution in sterile-deionized water.
    2. Drop 7 ml (per plate) of 10 µM DMBQ solution on the top of the roots of 1-week-old P. japonicum grown in vertical position in a square plate. Seal the plate with a surgical tape (or Parafilm).
    3. Cover the root part with aluminium foil.
    4. Place the plates horizontally in a growth chamber set at 26 °C under 16 h light and 8 h dark condition, keeping the root part covered with aluminium foil.
    5. Haustoria will be formed along the parasite roots within 24 h. Count haustorium number under the stereomicroscope (Figure 2).
      Note: P. japonicum roots are very sensitive to injury. If tissue damage is caused during the procedure, the percentage of formed haustorium might be altered.


      Figure 2. DMBQ-induced haustorium. A. Two-week-old P. japonicum growing in the absence of DMBQ; B. P. japonicum treated with 10 µM DMBQ; C and D. Enlarged pictures of (B). DMBQ-induced haustoria (pointed by white arrows) formed along P. japonicum roots. E to G. Transgenic P. japonicum roots photographs taken under confocal Leica TCS-SP5 II microscopy. A root transformed with the construct ProPjYUC3:3xVENUS-N7-Pro35S:RFP (Ishida et al., 2016) was observed under differential interference contrast filter and YFP filters. The merged picture (E) and the picture under YFP filter (F). Tissue carrying the construct CYCB1;2 pro::YFP was stained with 400 µg/ml propidium iodide to highlight root cell morphology. For details see the reference Ishida et al. (2011). Note that the haustoria developed in a rounded form with this method, which is distinct from the agar-based method described in Figure 1. The white bars represent 1 mm and the yellow bars 2.5 mm.

  5. Clarification of root tissues with chloral hydrate*
    1. Immerse the root tissues in chloral hydrate solution overnight at room temperature.
    2. To examine the haustorium under microscope, place them on glass slide soaked in choral hydrate solution with the slide cover on top.
    *Note: Chloral hydrate is a harmful reagent. Always use gloves to manipulate it and dispose it in an appropriate container.  

Data analysis

For accurate results we recommend to perform at least three independent experiments, using 10 to 15 plants in each repetition. The plants with injured roots should be excluded from the analysis.
Observe the presence or absence of the haustorium in individual plant under a stereomicroscope and calculate the percentage of plants with the haustorium. Alternatively, count the number of formed haustoria per plant and analyse the average data for each repetition.
In the case of transformed transgenic roots, inspect the presence of haustorium under a stereomicroscope and determine the percentage of individual transgenic root with parasitic organ. Calculate the average haustoria per plant for each biological experiment.

Notes

The injured root compromised their ability to develop haustorium in P. japonicum. Thus, only healthy roots should be considered for data analysis.

Recipes

  1. GM media
    1x Murashige and Skoog salts (Pre-mixed)
    1% (w/v) sucrose
    0.01% (w/v) Myo-inositol
    0.06% (w/v) 2-(N-morpholino)ethanesulfonic acid (MES) monohydrate
    0.8% (w/v) agar
    Adjust pH to 5.7 with 1 N KOH
    Sterilize it by autoclaving
    For each square plate 100 ml of GM media was poured
    The plates containing the media were stored at 4 °C for maximum 3-4 days
  2. DMBQ stock solution (10 mM)
    1. Dissolve 16.8 mg of 2,6-dimethoxy-1,4-benzoquinone in 10 ml of dimethyl sulfoxide (DMSO)
    2. Aliquot the DMBQ solution in 1.5 ml of sterilized plastic tube
    3. Protect from light by covering with aluminium foil
    4. Store under -20 °C until the moment of use
  3. Chloral hydrate solution
    8 g chloral hydrate
    1 ml glycerol
    2 ml deionized water
    Store at room temperature until the moment of use

Acknowledgments

This protocol was adapted from our published work (Ishida et al., 2016) and from (Albrecht et al., 1999). This work was supported by MEXT KAKENHI grants (Nos. 24228008 and 15H05959 to K.S., Nos. 25114521, 25711019 and 904 25128716 to S.Y.) and the Ph.D. fellowship programs (MEXT to JKI).

References

  1. Albrecht, H., Yoder, J. I. and Phillips, D. A. (1999). Flavonoids promote haustoria formation in the root parasite triphysaria versicolor. Plant Physiol 119(2): 585-592.
  2. Chang, M. and Lynn, D. G. (1986). The haustorium and the chemistry of host recognition in parasitic angiosperms. J Chem Ecol 12(2): 561-579.
  3. Cui, S., Wakatake, T., Hashimoto, K., Saucet, S. B., Toyooka, K., Yoshida, S. and Shirasu, K. (2016). Haustorial hairs are specialized root hairs that support parasitism in the facultative parasitic plant Phtheirospermum japonicum. Plant Physiol 170(3): 1492-1503.
  4. Ishida, J. K., Wakatake, T., Yoshida, S., Takebayashi, Y., Kasahara, H., Wafula, E., dePamphilis, C. W., Namba, S. and Shirasu, K. (2016). Local auxin biosynthesis mediated by a YUCCA flavin monooxygenase regulates haustorium development in the parasitic plant Phtheirospermum japonicum. Plant Cell 28(8): 1795-1814.
  5. Ishida, J. K., Yoshida, S., Ito, M., Namba, S. and Shirasu, K. (2011). Agrobacterium rhizogenes-mediated transformation of the parasitic plant Phtheirospermum japonicum. PLoS One 6(10): e25802.
  6. Saucet, S. B. and Shirasu, K. (2016). Molecular parasitic plant-host interactions. PLoS Pathog 12(12): e1005978.
  7. Spallek, T., Mutuku, M. and Shirasu, K. (2013). The genus striga: a witch profile. Mol Plant Pathol 14(9): 861-869.
  8. Westwood, J. H., Yoder, J. I., Timko, M. P. and dePamphilis, C. W. (2010). The evolution of parasitism in plants. Trends Plant Sci 15(4): 227-235.
  9. Yoshida, S., Cui, S., Ichihashi, Y. and Shirasu, K. (2016). The haustorium, a specialized invasive organ in parasitic plants. Annu Rev Plant Biol 67: 643-667.

简介

Phtheirospermum japonicum 是用作模型寄生植物的Orobanchaceae科的兼性根寄生植物。兼性根寄生虫在其根部的侧面部分形成称为吸管的侵入性器官。为了在功能上表征寄生能力,需要量化痰液数量。然而,这项任务是非常费力和耗时的。在这里,我们描述了一种有效的方案,通过引诱化学药物和宿主根系渗出物处理在体外诱导体外释放。血吸虫。

背景 寄生植物已经演变成从其他植物获得营养。一些寄生植物通过感染商业作物对农业造成重大损害(Spallek等人,2013)。有义务的寄生植物需要宿主完成其生命周期,而兼性寄生植物可以在没有宿主作为自养生物体的情况下存活,但如果宿主植物在附近则通过感染转移到异养(Westwood等人,2010)。所有寄生植物的共同特征是称为ium a的专门器官,其通过建立血管桥连接寄生虫与宿主(Saucet和Shirasu,2016; Yoshida等人,2016)。根尖寄生虫形成源于扩大根尖的终端诱捕物,而兼性根寄生虫形成侧向吸收,其在寄生虫根的侧面发育而不影响根分生组织。因此,可以在根部形成几种侧向吸收。淋巴细胞发育的早期特征是由细胞扩增和细胞分裂的组合引起的根部组织扩大。先前已经鉴定了几种能够诱导体外形成泡罩的宿主衍生物质。这些物质称为诱导诱导因子(HIF)。其中最活跃的HIF是DMBQ(2,6-二甲氧基-1,4-苯醌),最初从高粱根提取物中分离出来(Chang和Lynn,1986)。天竺葵属植物,它是研究寄生虫分子机制的理想模型,因为它的生命周期短,体积小,作为自交植物的简单遗传学(Ishida ,2011; Cui等人,2016)。另外,遗传操作 japonicum 现在是可行的(Ishida等人,2011),其大规模的转录组信息也是可用的(Ishida等人,2016)。在这里,我们报告了一种有效的体外方法来进行吸烟诱导,以研究P型烟碱相关基因的功能。血吸虫。该方法通过DMBQ处理或通过与宿主渗出物的接触提出了用于体外诱导诱导痰的方法。这种技术有助于了解触发寄生植物中ium trigger形成的遗传因素。

关键字:寄生, 寄生植物, 列当科, 吸器, 根, 宿主, 渗出物, DMBQ

材料和试剂

  1. Falcon 50ml圆锥形离心管(例如Corning,Falcon,sup。,目录号:352070)
  2. 滤纸,2号,Ø9厘米(Advantec,目录号:00021090)
  3. 直径为100mm的灭菌塑料盘皿(例如BioLiteφ100TC Dish,Fisher Scientific,目录号:12-556-002)
    制造商:Thermo Fisher Scientific,Thermo Scientific TM ,目录号:130182。
  4. Eppendorf 微量离心管(1.5ml)(例如,Fisher Scientific,目录号:05-408-129)
  5. 手术胶带(或石蜡膜)(例如,3M,目录号:1530-1)
  6. 厨房铝箔
  7. 灭菌的方形塑料盘皿140×100×14.5mm(Eiken Chemical,目录号:AW2000703077)
  8. 显微镜幻灯片(例如,Fisher Scientific,目录号:12-549-3)
  9. 显微镜盖玻片L x W x D:22 x 70 x 1.0 mm(例如,Fisher Scientific,目录号:10-016-24)
  10. 手套
  11. 水稻种子(日本粳稻日本晴)
  12. 日本。um种子
  13. 商业次氯酸盐溶液(Kao Japan)(约6%次氯酸钠)
  14. 氢氧化钾(KOH)(Sigma-Aldrich,目录号:484016)
  15. 吐温20(Sigma-Aldrich,目录号:P9416)
  16. 水(Milli-Q级)
  17. 碘化丙啶(Sigma-Aldrich,目录号:P4170)
  18. Murashige和Skoog盐(预混合)(Wako Pure chemical Industries,目录号:392-00591)
  19. 蔗糖(Merck Millipore,目录号:107687)
  20. 肌醇 - 肌醇(Sigma-Aldrich,目录号:I7508)
  21. 2,6-二甲氧基-1,4-苯醌(DMBQ)(Sigma-Aldrich,目录号:428566)
  22. 琼脂(Merck Millipore,目录号:101614)
  23. 二甲基亚砜(DMSO)(和光纯药,目录号:041-29351)
  24. 水合氯仿(Sigma-Aldrich,目录号:V000554)
  25. 甘油(Sigma-Aldrich,目录号:G5516)
  26. 通用媒体(见配方)
  27. DMBQ储备溶液(10 mM)(见配方)
  28. 水合氯醛溶液(见配方)

设备

  1. 稻壳(Fujiwara Scientific,型号:测试稻壳)
  2. 管旋转器(例如,TITEC,型号:RT-50,目录号:0000165-000)
  3. 层流罩(例如,YAMATO SCIENTIFIC,型号:CCV-1300E)
  4. 植物生长室(例如,NKsystem,型号:LPH-411SP)
  5. 日光白光荧光灯(NEC LIGHTING,型号:FL40SEX-N-HG)
  6. 显微镜(Leica Microsystems,型号:TCS-SP5 II)
  7. 涡旋振荡器(例如,Scientific Industries,型号:Vortex-Genie2,目录号:G560-SI-0246 2)
  8. 手术解剖刀手柄(例如,,Swann-Morton,目录号:0933)
  9. 手术刀片11号(例如,,Swann-Morton,目录号:0303)
  10. 不锈钢镊子(例如,Sigma-Aldrich,目录号:F4142-1EA)
  11. 半分析天平(例如,,Shimadzu,型号:AUW220D)
  12. 水浴(例如,,Fisher Scientific,型号:Fisher Scientific TM Isotemp TM 通用豪华水浴,目录号:S28124)
  13. 光立体显微镜(例如,卡尔·蔡司,型号:Stemi-2000)
  14. 光学显微镜(例如,奥林巴斯,型号:BX53-P)
  15. 高压灭菌器(例如,,平山,型号:HG系列)

程序

  1. 水稻种子发芽
    1. 用稻壳从水稻种子中取出种皮。
      注意:如果米糠不可用,可以通过用镊子或手紧紧捏住种子的and子并将其推下来逐一除去种皮。
    2. 将外套去除的种子放在50ml塑料管中。
      注意:每根管子最多可达20-30粒。
    3. 为了对水稻种子进行表面灭菌,将其浸入含0.1%(v/v)吐温20的50%(v/v)商业次氯酸盐溶液(最终溶液中约3%次氯酸钠)中。速度5分钟,将塑料管放置在管旋转器中25分钟,设定为10-20 rpm。
    4. 从这一步骤,程序应在无菌层流罩中进行,用无菌去离子水(Milli-Q级)将种子洗涤5次。
    5. 将表面灭菌的种子转移到放置在塑料培养皿(Ø100mm)中的一片潮湿的滤纸上。对于50粒种子,使用12-15毫升无菌水来润湿滤纸
    6. 将种子置于26℃,16小时光照条件下,8小时黑暗条件下的生长室中放置1周,其光照强度为约701摩尔/平方米/秒,由白光荧光灯(FL40SEX-N-FL,NEC,日本)

      图1.水稻根系分泌物处理 A.由解剖刀切除的一周龄水稻根部固定在琼脂块中。将琼脂块切成小块(〜2×4cm),每个包含约20-25mg切除的水稻根。它被放置在P上。日本植物保持垂直位置2周。 (A)放大图。 C.日本ium根无根诱导处理; D和E.沿着P形成的诱导的吸烟(由白色箭头指向)。日本根F.用水合氯醛溶液清洗根组织,并在显微镜下观察。 G和H.在转基因猪中形成的烟炱。在共聚焦徕卡TCS-SP5 II显微镜下拍摄合并过滤器(G)和YFP过滤器(H)照片的具有构建体的ProPjYUC3:3xVENUS-N7-Pro35S:RFP 。有关详细信息,请参见参考文献Ishida等人。 (2016)。白色条表示1厘米,黑色条纹为2.5毫米。

  2. P上。日本种子发芽
    1. 将种子放在1.5ml塑料管中(每个管最多约100个种子)
    2. 通过浸入10%(v/v)商业次氯酸盐溶液(〜0.6%次氯酸钠在最终溶液中)来灭菌种子表面。将管置于旋转振荡器中,最大速度设定1分钟。用新的漂白溶液替换溶液,并保持在旋转振荡器中9分钟。
    3. 从这个步骤,程序应该在无菌层流罩中进行,用无菌去离子水洗种子五次。
    4. 将种子浸入无菌去离子水中。将它们在4℃的黑暗中保持过夜。
    5. 使用1毫升的提示,在包含GM培养基的方形盘子的顶部逐一种植种子,每个板块约12-15个种子。用手术胶带密封方形板,并用铝箔盖住。保持种子在黑暗中三天以刺激发芽。
    6. 去除部分铝箔,保持根部的覆盖。
    7. 将正方形盘垂直放置在25℃,16小时光照和8小时黑暗条件下的生长室中2周。
      注意:让寄生植物垂直生长是很重要的,以避免根部渗入琼脂培养基。
  3. 水稻根系渗出物引起的。。
    1. 使用灭菌的镊子和解剖刀在灭菌的盘碟(Ø9厘米)上,从1周龄的水稻幼苗上切下根部。
    2. 测量根鲜重。对于180毫克鲜重的根组织,20-25切除根是必要的
    3. 在灭菌的盘碟(Ø9厘米)中,放置180毫克鲜重的水稻根和5毫升水,从水稻种子发芽的板上。
    4. 使用消毒的手术刀将根切成小块(约1-3毫米长)。
    5. 加入20毫升0.8%(w/v)琼脂,在60℃的水浴中加热
    6. 混合均匀,直到根部分布均匀分布在琼脂上
    7. 让琼脂固化30分钟。
    8. 将琼脂块切成大约〜2 x〜4厘米,并将它们放在生长在GM培养基上的寄生植物根上。琼脂块的大小应足以覆盖寄生根。 (图1A和1B)
      注意:当使用转基因根时,将它们转移到装有30ml含有抗生素头孢噻肟(300μg/ml)的0.8%(w/v)琼脂的平板皿(Ø9cm)中。含有切碎的水稻根和渗出物的琼脂块应放在转基因根上。
    9. 吸管将在24小时内形成(图1D至1F)。显示了形成ust ust的转基因根的实例(图1G至1H)
    10. 取出含有水稻根的琼脂,并计算立体显微镜下的吸烟数。
  4. 由DMBQ引起的呕吐物
    1. 将10mM DMBQ储备液稀释至10μMDMBQ工作溶液,在无菌去离子水中。
    2. 在1周龄的P的根部顶部滴下7ml(每片)10μMDMBQ溶液。日um um>。。。。。。。。。。。。用手术胶带(或Parafilm)密封板。
    3. 用铝箔覆盖根部。
    4. 将板在水平放置在26℃,16小时光照和8小时暗条件下的生长室中,保持根部分用铝箔覆盖。
    5. 在24小时内,沿着寄生虫根部形成鲈鱼。在立体显微镜下计数出汗数(图2)。
      注意:日本um根对损伤非常敏感。如果在手术过程中造成组织损伤,可能会改变形成的痰液的百分比。


      图2. DMBQ诱导的吸烟。 A.两周龄的

      日本; um>>;;;;;;;;;;;用10μMDMBQ处理B. 日本麻药 (B)放大照片。沿着P形成的DMBQ诱发的吸烟(由白色箭头指向)。日本根/转基因在共焦徕卡TCS-SP5 II显微镜下拍摄的日本照片。在差异干涉对比滤波器和YFP滤波器下观察到用构建体ProPjYUC3:3xVENUS-N7-Pro35S:RFP(Ishida等人,2016)转化的根。合并图片(E)和YFP滤镜(F)下的图片。携带构建体CYCB1; 2 pro :: YFP的组织用400μg/ml碘化丙啶染色以突出根细胞形态。详细信息参见Ishida等人。(2011)。注意,该方法以圆形形式发展,这与图1所示的基于琼脂的方法不同。白色条代表1mm,黄色条2.5mm。

  5. 用水合氯醛澄清根组织*
    1. 在水合氯醛水溶液中浸泡过夜,室温下浸泡过夜。
    2. 要在显微镜下检查痰液,将它们放在玻璃载玻片上,并将滑盖覆盖在顶部。
    注意:水合氯醛是一种有害的试剂。请务必使用手套进行操作并将其处理在相应的容器中。  

数据分析

为了获得准确的结果,我们建议至少进行三次独立实验,每次重复使用10到15个植物。受伤根的植物应从分析中排除。
在立体显微镜下观察个体植物中存在或不存在的烟草,并计算具有烟草的植物的百分比。或者,计算每个植物形成的吸烟数量,并分析每次重复的平均数据。
在转化的转基因根的情况下,检查在立体显微镜下的存在,并确定个体转基因根与寄生器官的百分比。计算每个生物实验的每株植物的平均吸烟量。

笔记

受伤的根部损害了他们在P中开发痰液的能力。血吸虫。因此,只有健康的根才应该考虑进行数据分析。

食谱

  1. 通用媒体
    1x Murashige和Skoog盐(预混)
    1%(w/v)蔗糖 0.01%(w/v)Myo - 肌醇
    0.06%(w/v)2-(N-吗啉代)乙磺酸(MES)一水合物
    0.8%(w/v)琼脂
    用1N KOH调节pH至5.7 通过高压消毒灭菌
    对于每个正方形板,将100ml转基因培养基倒入
    含有培养基的板在4℃下储存最多3-4天
  2. DMBQ储备溶液(10 mM)
    1. 将16.8mg 2,6-二甲氧基-1,4-苯醌溶于10ml二甲基亚砜(DMSO)中。
    2. 将DMBQ溶液等分于1.5ml灭菌塑料管中
    3. 用铝箔覆盖保护光线
    4. 存储在-20°C直到使用时刻
  3. 水合氯解决方案
    8克水合氯醛
    1毫升甘油
    2ml去离子水 在室温下存放直至使用时刻

致谢

该协议是从我们发表的作品(Ishida等人,2016)和(Albrecht等人,1999)中改编而成。这项工作得到了MEXT KAKENHI赠款(S.S.的编号24228008和15H05959,S.Y.的第25114521号,25711019号和90425128716号)和博士学位的支持。奖学金计划(MEXT to JKI)。

参考

  1. Albrecht,H.,Yoder,JI和Phillips,DA(1999)。黄酮类促进根寄生虫triphysaria versicolor中的诱饵形成。植物生理学119(2):585-592。
  2. Chang,M.and Lynn,DG(1986)。  寄生被子植物中的宿主和宿主识别的化学物质。 J Chem Ecol 12(2):561-579。
  3. Cui,S.,Wakatake,T.,Hashimoto,K.,Saucet,SB,Toyooka,K.,Yoshida,S。和Shirasu,K。(2016)。< a class ="ke-insertfile"href = "http://www.ncbi.nlm.nih.gov/pubmed/26712864"target ="_ blank">头发是专属的根毛,支持在兼性寄生植物中的寄生植物> um um um。。。。/a>植物生理 170(3):1492-1503。
  4. Ishida,JK,Wakatake,T.,Yoshida,S.,Takebayashi,Y.,Kasahara,H.,Wafula,E.,dePamphilis,CW,Namba,S。和Shirasu,K。(2016)。 class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/27385817"target ="_ blank">由YUCCA黄素单加氧酶介导的局部生长素生物合成调节寄生植物中的发酵 Phtheirospermum japonicum 植物细胞 28(8):1795-1814。
  5. Ishida,JK,Yoshida,S.,Ito,M.,Namba,S。和Shirasu,K。(2011)。发根土壤杆菌介导的寄生植物转化> um。。。。。。。。。。。。。。。。。。。。。。。。。。 ):e25802。
  6. Saucet,SB和Shirasu,K.(2016)。分子寄生植物 - 宿主相互作用。 PLoS Pathog 12(12):e1005978。
  7. Spallek,T.,Mutuku,M。和Shirasu,K。(2013)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/23841683"目标="_空白">属性striga:一个女巫的个人资料 Mol Plant Pathol 14(9):861-869。
  8. Westwood,JH,Yoder,JI,Timko,MP and dePamphilis,CW(2010)。  植物中寄生的演化。 趋势植物科学 15(4):227-235。
  9. Yoshida,S.,Cui,S.,Ichihashi,Y.和Shirasu,K。(2016)。寄生植物中的专门的侵入性器官的发酵物。 Annu Rev Plant Biol 67:643-667。
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引用:Ishida, J. K., Yoshida, S. and Shirasu, K. (2017). Haustorium Induction Assay of the Parasitic Plant Phtheirospermum japonicum. Bio-protocol 7(9): e2260. DOI: 10.21769/BioProtoc.2260.
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