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Pseudomonas syringae Flood-inoculation Method in Arabidopsis
拟南芥的丁香假单胞菌浸没接种法   

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参见作者原研究论文

本实验方案简略版
Molecular Plant Microbe Interactions
Feb 2016

Abstract

Pseudomonas syringae pv. tomato strain DC3000 (Pto DC3000), which causes bacterial speck disease of tomato, has been used as a model pathogen because of its pathogenicity on Arabidopsis thaliana. Here, we demonstrate a rapid and reliable flood-inoculation method based on young Arabidopsis seedlings grown on one-half strength MS medium. We also describe a method to evaluate the bacterial growth in Arabidopsis.

Keywords: Pseudomonas syringae (丁香假单胞菌), Arabidopsis thaliana (拟南芥), Plant innate immunity (植物天然免疫), Pathogenicity (致病性)

Background

The A. thaliana-Pto DC3000 model pathosystem is widely used to investigate the molecular mechanisms of microbial pathogenesis and plant innate immunity (Ishiga et al., 2012 and 2016; Ishiga and Ichinose, 2016). Although several inoculation methods have been developed to study the interactions in this model system, none of the methods reported to date are similar to those occurring in nature.

Materials and Reagents

  1. 1.5 ml microcentrifuge tubes (Ina-optika, catalog number: LT-0150 )
  2. 1 ml pipette tips (Mettler-Toledo, catalog number: 768/8 RT-1000 )
  3. Inoculating loops (VWR, catalog number: 12000-812 )
  4. 3M Micropore 2.5 cm surgical tape (3M, catalog number: 1530-1 )
  5. Plastic Petri plates (100 x 20 mm) (TrueLine, catalog number: TR4002 )
  6. Plastic Petri plates (100 x 15 mm) (VWR, catalog number: 10053-704 )
  7. Conical tubes (50 ml) (LabPlanet, catalog number: 3181-345-008 )
  8. Plant material: Arabidopsis thaliana ecotype Columbia (Col-0) was used as a wild-type plant
  9. Bacterial strains: P. syringae pv. tomato DC3000 (Pto DC3000) was used as pathogenic strain on A. thaliana-Pto DC3000 was kindly provided by Dr. Fumiaki Katagiri at University of Minnesota
  10. Ethanol (70%) (Wako Pure Chemical Industries, catalog number: 059-07895 )
  11. Sodium hypochlorite (Wako Pure Chemical Industries, catalog number: 197-02206 )
  12. Tween 20 (Wako Pure Chemical Industries, catalog number: 327-32475 )
  13. Sterile distilled water
  14. Rifampicin (50 mg/ml) (Wako Pure Chemical Industries, catalog number: 185-01003 )
  15. Silwet L-77 (BMS, catalog number: BMS-SL7755 )
  16. 5% H2O2 (Wako Pure Chemical Industries, catalog number: 081-04215 )
  17. Mannitol (Wako Pure Chemical Industries, catalog number: 133-00845 )
  18. L-glutamic acid (Wako Pure Chemical Industries, catalog number: 074-00505 )
  19. KH2PO4 (Wako Pure Chemical Industries, catalog number: 169-04245 )
  20. NaCl (Wako Pure Chemical Industries, catalog number: 191-01665 )
  21. MgSO4·7H2O (Wako Pure Chemical Industries, catalog number: 131-00405 )
  22. Agar (Wako Pure Chemical Industries, catalog number: 010-15815 )
  23. NaOH (Wako Pure Chemical Industries, catalog number: 192-15985 )
  24. Murashige and Skoog basal medium (Sigma-Aldrich, catalog number: M0404 )
  25. Sucrose (Wako Pure Chemical Industries, catalog number: 196-00015 )
  26. PhytagelTM (Sigma-Aldrich, catalog number: P8169 )
  27. KOH (Wako Pure Chemical Industries, catalog number: 165-21825 )
  28. Media
    1. Mannitol-glutamate (MG) medium (see Recipes)
    2. One-half strength Murashige and Skoog (MS) medium (see Recipes)

Equipment

  1. High-speed centrifuge (TOMY, model: MX305 )
  2. Pipette (Mettler-Toledo, model: Pipet-Lite XLS+ )
  3. Labo shaker (TAITEC, model: NR-2 )
  4. Labo incubator (AS ONE, model: ICV-300P )
  5. Clean work station (Hitachi Industrial Equipment Systems, model: CCV-1306E )
  6. Spectrophotometer (JASCO, model: V-630 )
  7. Plant growth chamber (NKsystem, model: LPH-411SP )
  8. Digital scale (AS ONE, model: IB-300 )
  9. Mortars and pestles (AS ONE, catalog number: 6-549-02 , φ90 mm)

Procedure

  1. Plant growth
    1. Incubate 100-200 Arabidopsis seeds in 70% ethanol for 5 min in a microcentrifuge tube.
    2. Allow the seeds to settle by centrifugation (500 x g for 30 sec) and remove 70% ethanol by pipetting.
    3. Incubate the seeds with 6% sodium hypochlorite containing 0.1% Tween 20 for 15 min with gentle shaking.
    4. Allow the seeds to settle by centrifugation (500 x g for 30 sec) and remove 6% sodium hypochlorite by pipetting.
    5. Rinse the seeds with sterile distilled water at least four times.
    6. Maintain the seeds in a small amount of sterile distilled water (300-500 μl).
      Note: The seeds can be stored at 4 °C. The cold treatment for 2 days improves the rate and synchrony of germination.
    7. Plant individual seeds in low density (50-80 seeds/plate) using a 1 ml pipette tip on one-half strength MS plates (Figure 1A and Video 1).
      Note: The one-half strength MS plates need to be dried overnight in a sterile hood work station with closed lids before transferring the surface-sterilized seeds. If the one-half strength MS plates are not dried enough, the vitreous and wet plants are observed. These plants are more sensitive to pathogen inoculation.


      Figure 1. Procedure of Pseudomonas syringae flood-inoculation method in Arabidopsis. A. Planting the seeds on one-half strength MS plates. B. Transferring the seedlings to new one-half strength MS plates at one week after germination with forceps. C. Incubation of the seedlings at 24 °C with a light intensity of 150-200 μE m-2 sec-1 and a 12 h light/12 h dark photoperiod. D. Two-week-old Arabidopsis seedlings for pathogen assays. E. Preparation of P. syringae pv. tomato DC3000 (Pto DC3000) inoculum. F. Flood-inoculation by dispensing the bacterial suspension onto the plates. G. Removal of the bacterial suspension by decantation. H. Disease phenotypes of Arabidopsis seedlings 4 days after inoculation with Pto DC3000.

      Video 1. Procedure of planting the seeds on one-half strength MS plates

    8. Incubate the seeds at 24 °C with a light intensity of 150-200 μE m-2 sec-1 and a 12 h light/12 h dark photoperiod for one week.
    9. Transfer the healthy seedlings at one-week post-germination to fresh new one-half strength MS plates with forceps (Figure 1B).
      Note: The roots need to be put on one-half strength MS plate without damaging and seven seedlings per plate are good for healthy plant growth.
    10. Incubate the seedlings at 24 °C with a light intensity of 150-200 μE m-2 sec-1 and a 12 h light/12 h dark photoperiod for one week (Figure 1C). Use the two-week old post-germination seedlings for the pathogen assays (Figure 1D).

  2. Bacterial growth and inoculum preparation
    1. Streak a -80 °C stock of bacterial culture on MG agar supplemented with rifampicin (50 μg/ml). Incubate the plate at 28 °C until bacterial colonies grow (24-48 h).
    2. Inoculate a single colony of Pto DC3000 onto new MG agar with rifampicin (50 μg/ml) and streak all over the plate. Incubate the plate at 28 °C (24-48 h).
    3. Collect as many bacterial colonies as possible with inoculating loop and suspend all colonies in an appropriate volume (20-30 ml) of sterile distilled water (Figure 1E).
      Note: Be careful not to take small pieces of MG agar medium.
    4. Measure the OD (A600) of the bacterial suspension and dilute to 4 x 106 CFU (colony forming units)/ml using sterile distilled water.
      Note: 1.0 OD measurement approximately corresponds to 2 x 108 CFU/ml.
    5. Add Silwet L-77 to a final concentration of 0.025% to the bacterial suspension and mix gently. Use the bacterial suspensions immediately after preparation.

  3. Flood-inoculation method
    1. Dispense 40 ml of bacterial suspension onto the plate containing the 2-week-old Arabidopsis seedlings and incubate for 2-3 min at room temperature (Figure 1F).
      Note: One bacterial plate normally allows to make more than 800 ml of bacterial suspension.
    2. Remove the bacterial suspension by decantation and seal the plates with 3M Micropore 2.5 cm surgical tape (Figure 1G).
    3. Incubate the inoculated seedlings at 24 °C with a light intensity of 150-200 μE m-2 sec-1 and a 12 h light/12 h dark photoperiod.
    4. Observe the symptom development at 2-4 days-post-inoculation (dpi) (Figure 1H).

  4. Evaluation of bacterial growth
    1. Collect four seedlings from a single plate by cutting the hypocotyls to separate the above agar parts (whole rosette) from the Phytagel plate at 0, 1, 2 and 3 dpi and measure the seedlings’ weight.
      Note: Avoid collecting vitreous and wet plants. These plants are more sensitive to pathogen inoculation.
    2. Surface-sterilize the seedlings in a beaker with 5% H2O2 for 3 min to determine the internal bacterial population and rinse the seedlings three times by dipping in sterile distilled water.
      Note: The bacterial populations at 0 dpi is estimated from seedlings collected 1 h post-inoculation without surface-sterilization by H2O2.
    3. Homogenize the seedlings completely with mortars and pestles for at least 1 min, and add 5 ml of sterile distilled water.
    4. Dilute the samples from 10-2 to 10-6 and plate on MG agar with rifampicin (50 μg/ml).
      Note: 10-2 to 10-3 dilution is suitable for 0 dpi and 10-4 to 10-6 is for 1, 2 and 3 dpi.
    5. Incubate the plates for 2-3 days and count the bacterial CFU using properly diluted samples.
      Note: Yellow non-mucoid colonies are observed after 2-3 days.
    6. Normalize the CFU as CFU/mg of tissue using the total weights of the inoculated seedlings.
      Note: The bacterial populations are evaluated from at least three biological replicates and each replicate represents a pooled sample of four independent seedlings from a single experiment grown in a single plate.

Data analysis

Student’s t-test was used for comparing two averages of bacterial growth, while ANOVA with post-hoc Tukey’s honest significant difference correction for multiple correction was applied when considering more than two averages of bacterial growth.

Recipes

  1. Mannitol-glutamate (MG) medium
    10 g mannitol
    2.0 g L-glutamic acid
    0.5 g KH2PO4
    0.2 g NaCl
    0.2 g MgSO4·7H2O
    15 g agar
    Adjust pH to 7.0 with 3 N NaOH and make up the volume to 1 L with distilled water prior to autoclaving
    Add rifampicin after autoclaving and pour MG medium into plastic Petri plates (100 x 15 mm)
  2. One-half strength Murashige and Skoog (MS) medium
    2.2 g Murashige and Skoog basal medium
    10 g sucrose
    3.0 g PhytagelTM (final 0.3%)
    Adjust pH to 5.8 with 1 N KOH and make up the volume to 1 L with distilled water prior to autoclaving
    Pour one-half strength MS medium into plastic Petri plates (100 x 20 mm)

Acknowledgments

This work was supported, in part, by the Program to Disseminate Tenure Tracking System, MEXT, Japan and JST ERATO NOMURA Microbial Community Control Project. This protocol was modified from previous work (Ishiga et al., 2011).

References

  1. Ishiga, Y. and Ichinose, Y. (2016). Pseudomonas syringae pv. tomato OxyR is required for virulence in tomato and Arabidopsis. Mol Plant Microbe Interact 29(2): 119-131.
  2. Ishiga, Y., Ishiga, T., Ikeda, Y., Matsuura, T. and Mysore, K. S. (2016). NADPH-dependent thioredoxin reductase C plays a role in nonhost disease resistance against Pseudomonas syringae pathogens by regulating chloroplast-generated reactive oxygen species. Peer J 4: e1938.
  3. Ishiga, Y., Ishiga, T., Uppalapati, S. R. and Mysore, K. S. (2011). Arabidopsis seedling flood-inoculation technique: a rapid and reliable assay for studying plant-bacterial interactions. Plant Methods 7: 32.
  4. Ishiga, Y., Ishiga, T., Wangdi, T., Mysore, K. S. and Uppalapati, S. R. (2012). NTRC and chloroplast-generated reactive oxygen species regulate Pseudomonas syringae pv. tomato disease development in tomato and Arabidopsis. Mol Plant Microbe Interact 25(3): 294-306.

简介

丁香假单胞菌 pv。 由于其在拟南芥中的致病性,已经将用于番茄细菌斑病的番茄株DC3000( DC3000)用作模型病原体 >。 在这里,我们展示了一种基于在一半强度MS培养基上生长的幼苗拟南芥幼苗的快速可靠的接种方法。 我们还描述了一种评估拟南芥细菌生长的方法。

背景 A。 DC3000模型病理学被广泛用于研究微生物发病机制和植物先天免疫的分子机制(Ishiga等,2012和2016; Ishiga和Ichinose,2016)。 虽然已经开发了几种接种方法来研究该模型系统中的相互作用,但迄今报道的方法之一与自然界中发生的方法相似。

关键字:丁香假单胞菌, 拟南芥, 植物天然免疫, 致病性

材料和试剂

  1. 1.5ml微量离心管(Ina-optika,目录号:LT-0150)
  2. 1 ml移液管吸头(Mettler-Toledo,目录号:768/8 RT-1000)
  3. 接种环路(VWR,目录号:12000-812)
  4. 3M Micropore 2.5厘米手术胶带(3M,目录号:1530-1)
  5. 塑料Petri板(100 x 20 mm)(TrueLine,目录号:TR4002)
  6. 塑料Petri板(100 x 15 mm)(VWR,目录号:10053-704)
  7. 锥形管(50ml)(LabPlanet,目录号:3181-345-008)
  8. 植物材料:拟南芥生态型哥伦比亚(Col-0)用作野生型植物
  9. 细菌菌株:丁香假单胞菌pv。将DC3000( DC3000)在番茄中用作致病菌株。 taliana - Pto DC3000由明尼苏达大学的Fumiaki Katagiri博士亲自提供
  10. 乙醇(70%)(Wako Pure Chemical Industries,目录号:059-07895)
  11. 次氯酸钠(Wako Pure Chemical Industries,目录号:197-02206)
  12. 吐温20(和光纯药,目录号:327-32475)
  13. 无菌蒸馏水
  14. 利福平(50mg/ml)(Wako Pure Chemical Industries,目录号:185-01003)
  15. Silwet L-77(BMS,目录号:BMS-SL7755)
  16. 5%H 2 O 2(和光纯药,目录号:081-04215)
  17. 甘露醇(和光纯药,目录号:133-00845)
  18. L-谷氨酸(Wako Pure Chemical Industries,目录号:074-00505)
  19. (和光纯药,目录号:169-04245)
  20. NaCl(Wako Pure Chemical Industries,目录号:191-01665)
  21. MgSO 4·7H 2 O(和光纯药,目录号:131-00405)
  22. 琼脂(和光纯药,目录号:010-15815)
  23. NaOH(和光纯药,目录号:192-15985)
  24. Murashige和Skoog基础培养基(Sigma-Aldrich,目录号:M0404)
  25. 蔗糖(和光纯药,目录号:196-00015)
  26. Phytagel TM (Sigma-Aldrich,目录号:P8169)
  27. KOH(和光纯药,目录号:165-21825)
  28. 媒体
    1. 甘露醇 - 谷氨酸(MG)培养基(参见食谱)
    2. 一半强度的Murashige和Skoog(MS)媒介(见配方)

设备

  1. 高速离心机(TOMY,型号:MX305)
  2. 移液器(Mettler-Toledo,型号:Pipet-Lite XLS +)
  3. Labo振动筛(TAITEC,型号:NR-2)
  4. Labo孵化器(AS ONE,型号:ICV-300P)
  5. 清洁工作站(日立工业设备系统,型号:CCV-1306E)
  6. 分光光度计(JASCO,型号:V-630)
  7. 植物生长室(NKsystem,型号:LPH-411SP)
  8. 数字秤(AS ONE,型号:IB-300)
  9. 砂浆和杵(AS ONE,目录号:6-549-02,φ90mm)

程序

  1. 植物生长
    1. 在微量离心管中将100-200μg拟南芥种子在70%乙醇中孵育5分钟。
    2. 通过离心(500×g×30秒)使种子沉降,并通过移液除去70%乙醇。
    3. 用含有0.1%吐温20的6%次氯酸钠孵育种子15分钟,轻轻摇动。
    4. 通过离心(500×g×30秒)使种子沉降,并通过移液除去6%次氯酸钠。
    5. 用无菌蒸馏水冲洗种子至少四次。
    6. 将种子保存在少量无菌蒸馏水(300-500μl)中 注意:种子可以在4°C储存。 2天的冷处理提高了萌发率和同步性
    7. 在半强度MS板上使用1毫升移液管针头(图1A和视频1),以低密度(50-80种子/板)种植单个种子。
      注意:一半强度的MS板需要在带有封闭盖子的无菌罩工作站中干燥过夜,然后转移表面灭菌的种子。如果一半强度的MS板没有足够干燥,则观察玻璃体和湿的植物。这些植物对病原体接种更为敏感

      图1. 拟南芥中的注射方法 A.在半强度MS板上种植种子。 B.用镊子萌发后一周将幼苗转移到新的一半强度MS板上。 C.在光照强度为150-200微米/秒的条件下孵育幼苗24小时和12小时光/12小时暗光周期。 D.用于病原体测定的两周龄拟南芥幼苗。 E.制备。丁香草 pv。 DC3000( DC3000)接种物。 F.通过将细菌悬浮液分配到板上来进行洪水接种。 G.通过倾析除去细菌悬浮液。 H.用P3 DC3000接种4天后,拟南芥幼苗的疾病表型。

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      视频1.在半强度MS板上种植种子的步骤
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    8. 将种子在24°C下孵育,光强度为150-200微米/秒/秒/小时,12小时光/12小时暗光周期孵育一周。
    9. 将萌发后一周的健康幼苗转移到新的具有镊子的新的一半强度的MS板上(图1B)。
      注意:根系需要放在一半强度的MS板上,不会损坏,每块板上有七棵幼苗对健康植物生长有益。
    10. 在24℃的温度下孵育幼苗,光强度为150-200μEm-2至 sec -1和12小时光/12小时暗光周期一周(图1C)。使用两周龄的后发芽幼苗进行病原体测定(图1D)
  2. 细菌生长和接种物准备
    1. 在补充有利福平(50μg/ml)的MG琼脂上条纹a -80℃的细菌培养物。在28℃孵育平板直到细菌菌落生长(24-48小时)。
    2. 将DC3000的单个菌落接种到具有利福平(50μg/ml)的新MG琼脂上,并在板上连续条纹。在28℃(24-48小时)孵育该板。
    3. 用接种环收集尽可能多的细菌菌落并将所有菌落悬浮在适当体积(20-30ml)的无菌蒸馏水中(图1E)。
      注意:注意不要取少量的MG琼脂培养基。
    4. 测量细菌悬浮液的OD(A600),并用无菌蒸馏水稀释至4×10 6 CFU(菌落形成单位)/ml。
      注意:1.0 OD测量大致对应于2×10 8/CFU/ml。
    5. 将Silwet L-77加入到细菌悬浮液的最终浓度为0.025%,并轻轻混合。准备后立即使用细菌悬浮液。

  3. 洪水接种方法
    1. 将40ml细菌悬浮液分配到含有2周龄拟南芥幼苗的板上,并在室温下孵育2-3分钟(图1F)。
      注意:一个细菌板通常允许制造超过800毫升的细菌悬浮液。
    2. 通过倾析除去细菌悬浮液,并用3M Micropore 2.5 cm手术胶带密封板(图1G)。
    3. 将接种的幼苗在24℃下孵育,光强度为150-200μE/秒/秒以上,12小时光/12小时暗光周期。
    4. 观察接种2-4天后的症状发展(dpi)(图1H)
  4. 细菌生长评估
    1. 通过切下下胚轴从单板收集四棵幼苗,以0,1,2和3dpi从Phytagel板分离上述琼脂部分(整个花环),并测量幼苗的重量。
      注意:避免收集玻璃体和湿的植物。这些植物对病原体接种更敏感
    2. 在具有5%H 2 O 2 O 2的烧杯中对幼苗进行表面灭菌3分钟以确定内部细菌群体并通过浸入无菌蒸馏水中冲洗幼苗三次。
      注意:在0dpi的细菌群体从接种后1小时收集的幼苗估计,没有通过H 2进行表面灭菌O 2
    3. 用砂浆和杵将幼苗均匀化至少1分钟,并加入5ml无菌蒸馏水。
    4. 将样品从10℃-2小时稀释至10℃-6℃,并用利福平(50μg/ml)在MG琼脂上铺板。
      注意:10
      -2 至10 -3 稀释适用于0 dpi和10 到10 /em> 适用于1,2和3 dpi。
    5. 孵育板2-3天,并使用适当的稀释样品计数细菌CFU。
      注意:2-3天后观察黄色非粘液样菌落
    6. 使用接种的幼苗的总重量将CFU归一化为CFU/mg组织。
      注意:从至少三个生物复制物评估细菌群体,每个复制品代表在单个板块中生长的单个实验中的四个独立幼苗的合并样品。

数据分析

使用Student's t -test来比较细菌生长的两个平均值,而考虑两次以上细菌生长平均值时,应用ANOVA与事后Tukey的多重校正的诚实显着差异校正。

食谱

  1. 甘露醇 - 谷氨酸(MG)培养基
    10g甘露醇
    2.0g L-谷氨酸
    0.5g KH PO 4
    0.2克NaCl
    0.2g MgSO 4·7H 2 O→// 15克琼脂
    用3N NaOH将pH调节至7.0,然后用蒸馏水将体积升至1升,然后进行高压灭菌 高压灭菌后加入利福平,将MG培养基倒入塑料培养皿(100 x 15 mm)中
  2. 一半强度的Murashige和Skoog(MS)媒介
    2.2克Murashige和Skoog基础培养基
    10g蔗糖
    3.0 g Phytagel TM (最终0.3%)
    用1N KOH调节pH至5.8,然后用蒸馏水将体积调节至1升,然后再进行高压灭菌 将一半强度的MS培养基倒入塑料培养皿(100 x 20毫米)

致谢

这项工作部分得到了传播任期跟踪系统,MEXT,日本和JST ERATO NOMURA微生物群落控制项目的支持。该协议是从以前的工作(Ishiga等人,2011)修改的。

参考文献

  1. Ishiga,Y。和Ichinose,Y。(2016)。 丁香假单胞菌 pv。 需要OxyR用于番茄和拟南芥中的毒力。 Mol Plant Microbe Interact 29(2):119-131。 br />
  2. Ishiga,Y.,Ishiga,T.,Ikeda,Y.,Matsuura,T.和Mysore,KS(2016)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm NADPH依赖型硫氧还蛋白还原酶C通过调节叶绿体产生的活性氧来对抗


    病原体的非宿主病抗性发挥作用。一个> 对等J 4:e1938。
  3. Ishiga,Y.,Ishiga,T.,Uppalapati,SR和Mysore,KS(2011)。 拟南芥种苗淹水技术:用于研究植物细菌相互作用的快速可靠的测定。 32.
  4. Ishiga,Y.,Ishiga,T.,Wangdi,T.,Mysore,KS和Uppalapati,SR(2012)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm。 NIRC和叶绿体生成的活性氧调节了丁香假单胞菌(Pseudomonas syringae),pv。(p.No.Gov/pubmed/22112219"target ="_ blank")。 番茄和中的番茄病发展。。 Mol Plant Microbe Interact 25(3):294-306。
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引用:Ishiga, Y., Ishiga, T., Ichinose, Y. and Mysore, K. S. (2017). Pseudomonas syringae Flood-inoculation Method in Arabidopsis. Bio-protocol 7(2): e2106. DOI: 10.21769/BioProtoc.2106.
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