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Assays to Assess Virulence of Xanthomonas axonopodis pv. manihotis on Cassava
木薯中地毯草黄单胞菌毒性的实验评估   

Megan CohnMegan Cohn*Mikel  ShybutMikel Shybut*Douglas DahlbeckDouglas DahlbeckBrian StaskawiczBrian Staskawicz  (*contributed equally to this work)
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Abstract

Cassava (Manihot esculenta) is a root crop that provides calories for people living in more than 100 tropical and subtropical countries and serves as a raw material for processing into starch and biofuels as well as feed for livestock (Howeler et al., 2013). Xanthomonas axonopodis pv. manihotis (Xam), the causal agent of cassava bacterial blight (CBB), can cause extensive crop damage (reviewed in Lopez et al., 2012; Lozano, 1986). Bacterial movement, growth in planta and the ability to cause disease symptoms are all important measures of bacterial fitness and plant susceptibility to CBB. Here we present a protocol for visualizing the movement of Xam within the plant. We also provide a detailed method of assaying bacterial growth in the cassava leaf midvein, and bacterial growth and disease symptom development in the leaf apoplast. These methods will be important tools for determining Xam strain pathogenicity and for developing cassava varieties that are resistant to CBB.

Keywords: Cassava(木薯), Xanthomonas axonopodis(地毯草黄单胞菌), Growth assay(生长测定), Bacteria(细菌), Plant disease(植物病害)

Materials and Reagents

  1. Cassava plants, cultivar TMS60444 (see Note 1)
  2. Xam strains (grown 48 h on NYGA plates + antibiotic selection)
  3. MgCl2
  4. Peptone (Thermo Fisher Scientific, catalog number: BP1420-500 )
  5. Yeast extract (Thermo Fisher Scientific, catalog number: BP1422-500 )
  6. Glycerine
  7. Agar (Thermo Fisher Scientific, catalog number: BP1423-500 )
  8. NYGA (see Recipes)

Equipment

  1. 28 °C incubator
  2. 5 ¾” glass disposable Pasteur pipet (Thermo Fisher Scientific, catalog number: 13-678-20B ), with the tip broken and filed to make a smooth 2 mm diameter tip (Figure 3A)
  3. 2 in. x 4 in. cardboard covered with Parafilm
  4. Mini-Beadbeater-96 (Biospec Products, catalog number: 1001 )
  5. 3 mm glass beads (Thermo Fisher Scientific, catalog number: 11-312A )
  6. 100 x 15 mm petri dishes
  7. Spectrophotometer (Pharmacia Biotech Ultrospec 3000 )
  8. Approximately 0.6 cm2 cork borer
  9. 1.7 ml Posi-Click tubes (Denville, catalog number: C-2170 )
  10. Single edge razor blade (Garvey Products Inc., catalog number: 40475 )
  11. 1 ml needleless syringe (BD, catalog number: 309659 )
  12. 96 well plate reader for measuring luminescence (PerkinElmer, catalog number: 2104-0010 )
  13. 96 well OptiPlate, black (PerkinElmer, catalog number: 6005270 )
  14. Dark room, x-ray imaging cassettes with film, ring stand

Procedure

Part I. Visualization of Xam movement

  1. Preparation of bioluminescent bacteria
    1. Make and transform competent Xam cells [as in Do Amaral et al. (2005)] with an expression vector containing the lux operon genes luxCDABEG from Vibrio fischeri (pLAFR-lux, unpublished, see acknowledgments).
    2. Successful transformants may be screened for luciferase expression using an EnVision microplate reader and the US LUM 96 default protocol before inoculation into cassava.
      1. Grow transformed Xam colonies at 28 °C for 2 days on NYGA plates containing the appropriate antibiotics (Rifampicin 100 µg/ml, Tetracycline 10 µg/ml).
      2. Resuspend Xam in 10 mM MgCl2 at OD600 = 0.6.
      3. Add 100 µl to each well of a 96 well OptiPlate, including negative controls (10 mM MgCl2 alone, untransformed Xam) as well as a positive control (E. coli with pLAFR-lux).
      4. Run the US LUM 96 protocol and read the output for each well. Look for samples with values equal to or greater than the positive control.

  2. Inoculations
    1. Two days prior to the inoculation, streak out the Xam strains to be used from a glycerol stock onto NYGA plates containing the appropriate antibiotics and place at 28 °C for 2 days.
    2. Prepare an inoculum from this plate by suspending the bacteria at OD600 = 0.01 in 1 ml 10 mM MgCl2 (see Note 2).
    3. On the abaxial side of a leaf, create 2-3 close, shallow nicks with the tip of a razor blade, avoiding major veins (Figure 1A).
    4. Use a 1 ml needleless syringe to inoculate the bacterial suspension (OD600 = 0.01) into the leaf via the nicks to a total area of approximately 0.25 cm2. Using a finger on the free hand, push the leaf against the syringe while infiltrating, applying enough pressure to encourage the inoculum to enter the leaf (Figure 1B). Alternatively, a toothpick dipped in the Xam (pLAFR-lux) inoculum may be used to inoculate by directly puncturing the leaf through the midvein or apoplast, though results may vary more than with apoplast infiltrations.


      Figure 1. Apoplast inoculations and sample processing. A. Cassava leaf with 3 shallow abaxial nicks made by a razor blade. B. Demonstration of leaf infiltration using needleless syringe. C. Picture showing bacterial suspension inoculated into the leaf apoplast with a needleless syringe. D. Diagram showing sampled area of inoculated leaf apoplast for growth assay 6 days post inoculation (dpi).

  3. Visualization
    1. After 5-6 days of growth at ambient temperature, the bacteria may be visualized by exposing the leaf directly to x-ray film (Figure 2). For a one-time visualization, leaves may be removed and imaged in a standard film cassette for about 1 h. For continued tracking of bacterial movement in the same leaf, place the plant in a dark room and use binder clips to sandwich the leaf and a sheet of x-ray film between two pieces of cardboard. Keep the leaf elevated using a ring stand with a platform. After 1 h, take off the clips, image the film, and then return the plant to its growth room intact for subsequent imaging.


      Figure 2. Visualization of Xam vascular movement in cassava by a luciferase reporter construct after inoculation into the apoplast. A. Examples of syringe infiltrated leaves (OD600 = 0.01) showing Xam entry into nearby veins and into the midvein. B. A toothpick inoculated leaf (OD600 = 0.01). C. A diseased leaf inoculated via toothpick puncture and its Xam profile. Signal loss is often observed at the site of inoculation most likely due to leaf tissue wilting. (X = inoculation point; DPI = days post inoculation).

Part II. Assaying Xam growth and symptom development

  1. Culture growth
    Grow Xam strains on NYGA plates + antibiotic selection for 40-48 h in a 28 °C incubator.

  2. Preparation of inoculum
    1. Resuspend plated bacteria in 1 ml of 10 mM MgCl2 at approximately OD600 = 1.0 for each strain. Dilute with 10 mM MgCl2 to OD600 = 0.2 for leaf midvein growth assays, OD600 = 0.00005 (first to OD600 = 0.01, then to final OD) for apoplast growth assays, or OD600 = 0.01 for apoplast symptom assays.
    2. Select and label cassava leaves to inoculate (see Note 3). Leaves should be labeled with a marker to indicate the inoculated bacterial strain, timepoint, and replicate number (see Note 4).

  3. Inoculation

    Leaf midvein growth assay
    1. Hold the leaf on a piece of Parafilm-covered cardboard, adaxial side up.
    2. Dip the 2 mm diameter Pasteur pipet tip in 10 mM MgCl2 and then use it to punch a clean hole through the midvein approximately 4-5 cm in from the leaf tip. Make sure that a film of 10 mM MgCl2 stays in the hole to prevent air from entering the midvein as this will block uptake of the bacterial suspension (Figure 3A-B).
    3. Immediately pipette a 5 µl drop of OD600 = 0.2 bacterial suspension on the hole (Figure 3C). Move on to the next inoculation point and repeat. Let the drops dry completely without being disturbed. This can take up to 30 min.
    4. When all inoculations are complete and dry, begin tissue extraction for Day 0.


      Figure 3. Midvein growth assay inoculations and sample processing. A. A glass Pasteur pipet is modified to make an approximately 2 mm hole in the leaf midvein. B. Midvein inoculation point showing film of 10 mM MgCl2 after puncture. C. Midvein inoculation point with 5 µl drop of bacterial suspension. D. A cassava leaf with midvein inoculations on the middle 3 leaflets. A 0.6 cm2 disc including the inoculation point is taken at days 0 and 6 (dashed lines).

    Leaf apoplast growth and symptom assays
    1. On the abaxial side of a leaf, create 2-3 close, shallow nicks with the tip of a razor blade, avoiding major veins (Figure 1A).
    2. Use a 1 ml needleless syringe to inoculate the bacterial suspension (OD600 = 0.00005 for growth assays and OD600 = 0.01 for symptom assays) into the leaf via the nicks to a total area of approximately 0.25 cm2. To do this, use a finger on the free hand, push the leaf against the syringe while infiltrating, applying enough pressure to encourage the inoculum to enter the leaf (Figure 1B).
    3. For symptom assays, observe each site daily and keep track of the appearance and severity of water soaking symptoms, often observed by 6 days post inoculation (Figure 4).


      Figure 4. Representative examples of Xam water soaking symptoms. ++ indicates high levels of water soaking, + indicates medium water soaking, +/- indicates little or no water soaking.

  4. Tissue extraction for growth assays

    Leaf midvein growth assay (see Note 5)
    1. Using a cork borer, take an approximately 0.6 cm2 round leaf punch with the inoculation point exactly in the middle (Figure 3D). Place the punch in a Posi-Click tube containing a 3 mm glass bead and 200 µl 10 mM MgCl2.
    2. Pulverize the tissue using a bead beater (2 min., 36 oscillations/second). Bring each sample to a volume of 1 ml by adding 800 µl 10 mM MgCl2.

    Leaf apoplast growth assay
    1. Using a cork borer, take an approximately 0.6 cm2 round leaf punch with the inoculation site exactly in the middle (Figure 1D). Place the punch in a Posi-Click tube containing a 3 mm glass bead and 200 µl 10 mM MgCl2.
    2. Pulverize the tissue using a bead beater (2 min, 36 oscillations/second). Bring each sample to a volume of 1 ml by adding 800 µl 10 mM MgCl2.

  5. Dilutions and plating
    1. Make serial dilutions of the 1 ml sample of pulverized tissue in 10 mM MgCl2 and plate countable colony forming units (CFUs) (Figure 5A).


      Figure 5. Plating and counting Colony Forming Units (CFUs). A. Diagram of plated samples showing serial dilutions. B. Chart showing representative results of an apoplast growth assay [previously published in Cohn et al. (2014)].

      Leaf midvein growth assay
      1. Inoculation point (day 0): Make 1 ml 10-2 and 10-4 dilutions in microcentrifuge tubes (see Note 6). Plate fractions of these dilutions to make final dilutions of 10-4, 10-5, and 10-6 (see Note 7).
      2. Inoculation point (day 6): Make 1 ml 10-4 and 10-6 dilutions in microcentrifuge tubes. Plate fractions of these dilutions to make final dilutions of 10-5, 10-6, and 10-7 (see Note 8).

      Leaf apoplast growth assay
      1. Inoculation site (day 0): Plate 100 µl of the pulverized tissue sample for a 10-1 dilution.
      2. Inoculation site (day 6): Make 1 ml 10-2 and 10-4 dilutions in microcentrifuge tubes. Plate fractions of these dilutions to make final dilutions of 10-4, 10-5, and 10-6.
    2. Place plates in a 28 °C incubator for 4 days.
    3. Count colonies and correct for dilutions. For example, 5 colonies on the 10-6 dilution plate translates to 5,000,000 CFUs present in the original sample.
    4. Convert to log10 CFUs per portion of leaf sampled and plot (Figure 5B).

Notes

  1. Plants should be 2-3 months old. Our plants are propagated through cuttings (Note 9) and grown in a greenhouse room with no artificial lighting, mist (schedule: 15 min on/15 min off between 6:00 am and 6:00 pm) and an average temperature of 27 °C.
  2. An OD600 of 0.1 is approximately 108 colony forming units (CFU) per ml.
  3. Careful leaf selection is very important to cut down on technical variation. Only the first 2 fully unfolded leaves should be used. It is best to use leaflets that are similar in size.
  4. We typically do 6 replicates per strain per timepoint. An average experiment will have 6 strains to be tested at days 0 and 6, so with 2 timepoints and 6 replicates per strain, that makes 72 inoculation points.
  5. In addition to assaying midvein growth at the inoculation point, one can also measure growth in the proximal midvein section, though this technique is more technically challenging and leaf choice becomes very important in order to cut down on sample variation (see Note 3). To measure bacterial growth in the proximal midvein section, use a razor blade to cut out the 3 cm of leaf midvein directly above the leaf punch encompassing the inoculation point at day 6. Place the midvein section in a Posi-Click tube containing a 3 mm glass bead and 200 µl 10 mM MgCl2. Pulverize the tissue using a bead beater (2 min, 36 oscillations/second). Bring each sample to a volume of 1 ml by adding 800 µl 10 mM MgCl2. Make 1 ml 10-2 and 10-4 dilutions in microcentrifuge tubes (see Note 6). Plate fractions of these dilutions to make final dilutions of 10-4, 10-5, and 10-6 (see Note 7).
  6. For example, to make a 10-2 dilution, add 10 µl of the pulverized tissue suspension to 990 µl of 10 mM MgCl2.
  7. For example, to plate a 10-6 dilution, put 10 µl of a 10-4 dilution on a plate and spread; to plate a 10-7 dilution, put 100 µl of a 10-6 dilution on a plate and spread.
  8. These dilutions are guidelines as the actual dilutions will depend on the virulence of the Xam strains that you are testing. The dilutions should be determined based on what will allow you to clearly count colonies after plating.
  9. To propagate plants through cuttings, cut all the leaves off of a plant that is woody along at least half of the stem (6-12 months old). Cut the stem into sections, making sure to include 3-4 nodes per section. Place each section into fertilized soil, taking care to maintain up/down orientation of the stem section and making sure that at least one node is below the soil surface, and at least one node is above the soil surface. Provide the cutting plenty of water. New leaves should begin growing from the top node after about a week.

Recipes

  1. NYGA (1 L)
    5 g peptone
    3 g yeast extract
    20 ml 100% glycerine
    1.5 g agar
    1. Combine peptone, yeast extract, and glycerine, then bring to 1 L with water, mixing on low heat.
    2. Bring pH to 7.0 using 1 M NaOH.
    3. Mix in agar.
    4. Autoclave 30 min.

Acknowledgments

This work was funded by NSF/BREAD (grant 0965418, BJS), NSF Graduate Research Fellowship (MC, MS), and a NIH Genetics Training Grant 2T32GM007127-36A1 (MC). pLAFR-lux vector was provided by Sebastian Schornack via Frank Thieme. Thank you to Rose Kantor for assisting MS with the inoculations seen in Figure 1. The midvein growth assay protocol was adapted from Castiblanco et al. (2013).

References

  1. Castiblanco, L. F., Gil, J., Rojas, A., Osorio, D., Gutierrez, S., Munoz-Bodnar, A., Perez-Quintero, A. L., Koebnik, R., Szurek, B., Lopez, C., Restrepo, S., Verdier, V. and Bernal, A. J. (2013). TALE1 from Xanthomonas axonopodis pv. manihotis acts as a transcriptional activator in plant cells and is important for pathogenicity in cassava plants. Mol Plant Pathol 14(1): 84-95.
  2. Cohn, M., Bart, R. S., Shybut, M., Dahlbeck, D., Gomez, M., Morbitzer, R., Hou, B. H., Frommer, W. B., Lahaye, T. and Staskawicz, B. J. (2014). Xanthomonas axonopodis virulence is promoted by a transcription activator-like effector-mediated induction of a SWEET sugar transporter in cassava. Mol Plant Microbe Interact 27(11): 1186-1198.
  3. Do Amaral, A. M., Toledo, C. P., Baptista, J. C. and Machado, M. A. (2005). Transformation of Xanthomonas axonopodis pv. citri by electroporation. Fitopatologia Brasileira 30: 292-294.
  4. Howeler, R., Lutaladio, N. and Thomas, G. (2013). Save and Grow: Cassava: a guide to sustainable production intensification. Food and Agriculture Organization of the United Nations.
  5. López, C. E. and Bernal, A. J. (2012). Cassava bacterial blight: using genomics for the elucidation and management of an old problem. Trop Plant Biol 5(1): 117-126.
  6. Lozano, J. C. (1986). Cassava bacterial blight: a manageable disease. Plant Dis 70: 1089-1093.

简介

木薯( Manihot esculenta )是一种根茎作物,为生活在100多个热带和亚热带国家的人们提供热量,并且作为加工成淀粉和生物燃料以及牲畜饲料的原料(Howeler et al。,2013)。 Xanthomonas axonopodis pv。木薯细菌枯萎病(CBB)的致病因子可引起广泛的作物损害(参见Lopez等人,2012; Lozano,1986)。细菌运动,植物中的生长和引起疾病症状的能力都是细菌适合性和植物对CBB敏感性的重要测量。在这里,我们提出了一个用于可视化植物中的Xam 运动的协议。我们还提供了一种详细的方法来检测木薯叶中期的细菌生长,以及叶片外质层中的细菌生长和疾病症状发展。这些方法将是确定Xam 菌株致病性和开发对CBB有抗性的木薯品种的重要工具。

关键字:木薯, 地毯草黄单胞菌, 生长测定, 细菌, 植物病害

材料和试剂

  1. 木薯植物,品种TMS60444(见注1)
  2. 菌株(在NYGA平板上生长48小时+抗生素选择)
  3. MgCl 2
  4. 蛋白胨(Thermo Fisher Scientific,目录号:BP1420-500)
  5. 酵母提取物(Thermo Fisher Scientific,目录号:BP1422-500)
  6. 甘油
  7. 琼脂(Thermo Fisher Scientific,目录号:BP1423-500)
  8. NYGA(见配方)

设备

  1. 28℃培养箱
  2. 5¾"玻璃一次性巴斯德吸管(Thermo Fisher Scientific,目录号:13-678-20B),使尖端破碎并锉成平滑的直径为2mm的尖端(图3A)
  3. 2英寸×4英寸的覆盖有石蜡膜的纸板
  4. Mini-Beadbeater-96(Biospec Products,目录号:1001)
  5. 3mm玻璃珠(Thermo Fisher Scientific,目录号:11-312A)
  6. 100×15mm培养皿
  7. 分光光度计(Pharmacia Biotech Ultrospec 3000)
  8. 约0.6厘米2 软木钻孔器
  9. 1.7ml Posi-Click管(Denville,目录号:C-2170)
  10. 单刃剃刀刀片(Garvey Products Inc.,目录号:40475)
  11. 1ml无针注射器(BD,目录号:309659)
  12. 用于测量发光的96孔板读数器(PerkinElmer,目录号:2104-0010)
  13. 96孔OptiPlate,黑色(PerkinElmer,目录号:6005270)
  14. 暗室,带胶片的X光成像暗盒,环形支架
  • 1.7ml Posi-Click管(Denville,目录号:C-2170)
  • 单刃剃刀刀片(Garvey Products Inc.,目录号:40475)
  • 1ml无针注射器(BD,目录号:309659)
  • 用于测量发光的96孔板读数器(PerkinElmer,目录号:2104-0010)
  • 96孔OptiPlate,黑色(PerkinElmer,目录号:6005270)
  • 暗室,带胶片的X光成像暗盒,环形支架
  • ... EnVision microplate reader and the US LUM 96 default protocol before inoculation into cassava.
    1. Grow transformed Xam colonies at 28 °C for 2 days on NYGA plates containing the appropriate antibiotics (Rifampicin 100 µg/ml, Tetracycline 10 µg/ml).
    2. Resuspend Xam in 10 mM MgCl2 at OD600 = 0.6.
    3. Add 100 µl to each well of a 96 well OptiPlate, including negative controls (10 mM MgCl2 alone, untransformed Xam) as well as a positive control (E. coli with pLAFR-lux).
    4. Run the US LUM 96 protocol and read the output for each well. Look for samples with values equal to or greater than the positive control.

  • Inoculations
    1. Two days prior to the inoculation, streak out the Xam strains to be used from a glycerol stock onto NYGA plates containing the appropriate antibiotics and place at 28 °C for 2 days.
    2. Prepare an inoculum from this plate by suspending the bacteria at OD600 = 0.01 in 1 ml 10 mM MgCl2 (see Note 2).
    3. On the abaxial side of a leaf, create 2-3 close, shallow nicks with 剃刀刀片的尖端,避免主要静脉(图1A)
    4. 用一个  1ml无针注射器接种细菌悬浮液(OD 600 = 0.01)通过切口进入叶,总面积约为0.25 cm 2。用手指在自由手上,将叶片推向注射器 同时渗透,施加足够的压力以鼓励接种物 进入叶片(图1B)。或者,浸泡在Xam(pLAFR-emlux)接种物中的牙签可以用于通过直接穿刺接种  叶通过midvein或apoplast,虽然结果可能变化更多 比外质体浸润

      图1.质外接种和样品处理 A.木薯叶 具有由剃刀刀片制成的3个浅的下轴切口。 B.示范 叶浸润使用无针注射器。 C.图片显示细菌  悬浮液用无针注射器接种到叶外质体中。  D.显示用于生长的接种的叶外质体的取样面积的图 测定接种后6天(dpi)

  • 可视化
    1. 后   在环境温度下生长5-6天,细菌可以是 通过将叶子直接暴露于x射线胶片而可视化(图2)。 为一个   一次可视化,叶可以被移除并在标准中成像 膜暗盒约1小时。 用于继续跟踪细菌 运动在同一片叶子,将植物放在黑暗的房间和使用粘合剂   夹子夹住叶子和一片X射线胶片在两片之间   的纸板。 保持叶子升高使用环架与a 平台。 1小时后,取下夹子,拍摄影片,然后返回   植物到其生长室完好以用于随后的成像

      图2.通过a。可视化木薯中的xam 血管运动 荧光素酶报道基因构建体接种到质外体中。 A. 注射器渗透的叶子(OD <600> = 0.01)的实例显示了Xam 条目 进入附近的脉和进入中脉。 B.牙签接种叶 (OD 600 = 0.01)。 C.通过牙签穿刺接种的病叶   其 Xam 配置文件。 信号丢失经常在现场观察到 接种最可能是由于叶组织枯萎。 (X =接种 点; DPI =接种后天数)。

  • 第二部分 测定Xam 生长和症状发展

    1. 文化发展
      在NYGA平板上生长 Xam 菌株,在28℃培养箱中抗生素选择40-48小时。

    2. 接种物的制备
      1. 在约1ml的10mM MgCl 2中重悬平板细菌 OD 600 = 1.0。 用10mM MgCl 2稀释至OD 600 = 0.2 叶中叶生长测定,OD 600 = 0.00005(首先OD 600 = 0.01,然后   至最终OD)用于质外体生长测定,或OD 600 = 0.01用于质外体 症状测定。
      2. 选择和标记木薯叶接种(见   注3)。 叶子应该用一个标记来标明 接种的细菌菌株,时间点和重复数(参见注释 4)。

    3. 联系

      叶中叶生长测定
      1. 把叶子放在一块石蜡膜覆盖的纸板上,近轴面朝上。
      2. 将2mm直径的巴斯德移液管尖端浸入10mM MgCl 2中,然后使用 它穿过一个清洁的洞穿过中间约4-5厘米 从叶尖。 确保10mM MgCl 2的膜保留在中 以防止空气进入中间层,因为这将阻挡吸收 的细菌悬浮液(图3A-B)。
      3. 立即移液a   将5μl滴在孔上的OD 600 = 0.2细菌悬浮液(图3C)。 移动到下一个接种点并重复。 让滴干 完全不受干扰。 这可能需要30分钟。
      4. 当所有接种完全和干燥时,开始组织提取第0天。


        图3.中脉生长测定接种和样品处理   玻璃巴斯德吸管被修改,以使一个大约2毫米的孔 叶中脉。 B.中膜接种点显示10mM的膜 穿刺后的MgCl 2。 C.中滴定接种点用5μl滴 细菌悬浮液。 D.在中期接种的木薯叶   中间3个传单。 包括接种点的0.6cm 2盘 在第0天和第6天(虚线)。

      叶质外质层生长和症状测定
      1. 在叶的背面,创造2-3关闭,浅刻痕 剃刀刀片的尖端,避免主要静脉(图1A)
      2. 用一个   1ml无针注射器接种细菌悬浮液(OD 600 = 对于生长测定为0.00005,对于症状测定为OD 600 = 0.01) 叶通过切口到约0.25cm 2的总面积。 去做 这个,用手指在自由的手上,推叶子靠着注射器 同时渗透,施加足够的压力以鼓励接种物 进入叶片(图1B)。
      3. 对于症状测定,观察每个 现场每天并跟踪水的外观和严重性 浸泡症状,通常在接种后6天观察到(图4)。


        图4.代表 Xam 水浸症状的实例。 ++表示高水平 水浸泡,+表示中等水浸泡,+/-表示少量或   无水浸泡。

    4. 生长测定的组织提取

      叶中叶生长测定(见注5)
      1. 使用软木钻孔器,取大约0.6cm 2的圆叶冲头 接种点恰好在中间(图3D)。 放置 在含有3mm玻璃珠和200μl10mM的Posi-Click管中冲孔   MgCl 2。
      2. 使用珠磨机(2分钟,36 振荡/秒)。 通过加入使每个样品达到1ml的体积 800μl10mM MgCl 2。

      叶质外质层生长测定
      1. 使用a 软木钻孔器,取大约0.6cm 2的圆叶冲头 接种位点正好在中间(图1D)。 将冲头放入   Posi-Click管,其含有3mm玻璃珠和200μl10mM MgCl 2。
      2. 使用珠磨机(2分钟,36 振荡/秒)。 通过加入使每个样品达到1ml的体积 800μl10mM MgCl 2。

    5. 稀释和电镀
      1. 使串行   稀释在10mM MgCl 2和10mM MgCl 2中的1ml粉碎组织样品 板计数菌落形成单位(CFU)(图5A)

        图5。 电镀和计数菌落形成单位(CFU)。 A.电镀图 样品显示连续稀释。 B.图表显示代表性 质外体生长测定的结果[以前在Cohn等人(2014)中发表]。

        叶中叶生长测定
        1. 接种点 (第0天):在微量离心管中制备1ml 10 -2和10μL稀释液(参见   注6)。 将这些稀释液的板级分进行最终稀释 10 -4 ,10 -5 和10 -6 (见注7)。
        2. 接种点(第6天): 在微量离心管中制备1ml 10 -4和10 -6 - 稀释。 盘子 这些稀释液的部分,以制备10μM,10μM,10μM和10μM的最终稀释物,和 10 -7 (见注8)。

        叶质外质层生长测定
        1. 接种部位(第0天):将100μl用于10 -1稀释的粉碎的组织样品平板。
        2. 接种位点(第6天):制备1ml 10 -2个和10个稀释于<! - SIPO -4 ,10 -5 和10 -6 的稀释液。
      2. 将板置于28℃培养箱中4天
      3. 计数菌落并校正稀释度。 例如,5个殖民地   10 -6 稀释板翻译成500,000 CFUs存在于 原始样品。
      4. 转化为每10份叶取样的log 10 CFU(图5B)。

    笔记

    1. 植物应为2-3个月大。 我们的植物通过插条繁殖(注9),并在没有人工照明,雾气(时间表:在上午6:00和下午6:00之间打开15分钟/15分钟)和平均温度27° C。
    2. 0.1的OD 600约为每ml约10 8个菌落形成单位(CFU)。
    3. 仔细选择叶片对减少技术变化非常重要。 只有前2个完全展开的叶片应该使用。 最好使用尺寸类似的传单。
    4. 我们通常每个时间点每个菌株进行6次重复。平均实验将在第0天和第6天测试6株,因此每个菌株有2个时间点和6个重复,使得72个接种点。
    5. 除了在接种点测定中脉生长外,还可以测量近端中脉部分的生长,尽管这种技术在技术上更具挑战性,并且叶选择变得非常重要,以减少样品变化(见注3)。为了测量近端中脉切片中的细菌生长,使用剃刀刀片在第6天时在包括接种点的叶冲头正上方切出3cm的叶中部。将中部切片置于含有3mm的Posi-Click管中玻璃珠和200μl10mM MgCl 2。使用珠磨机(2分钟,36振荡/秒)粉碎组织。通过加入800μl10mM MgCl 2使每个样品达到1ml的体积。在微量离心管中制备1ml 10 -2和10μL稀释液(见注6)。将这些稀释液的板级分制成10μM,10μM,10μM,10μM和10μMsup-6的最终稀释物(参见注释7)。 br />
    6. 例如,为了进行10μL稀释,将10μl粉碎的组织悬浮液加入990μl10mM MgCl 2溶液中。
    7. 例如,为了平板化10×10 -6稀释物,将10μL10×10 -4稀释物铺板在平板上并涂布;以铺板10 -7稀释,将100μl的10 < - > -6稀释物铺在板上并铺开。
    8. 这些稀释是指导,因为实际稀释将取决于正在测试的Xam 毒株的毒力。稀释应根据什么将允许您明确计数确定 电镀后的菌落
    9. 为了通过插条繁殖植物,将所有叶子切离沿至少一半茎(6-12个月龄)木质化的植物。 将茎切成部分,确保每个部分包括3-4个节点。 将每个部分放入受精土壤中,注意保持茎部的上/下取向,并确保至少一个节点在土壤表面下方,并且至少一个节点在土壤表面上方。 提供切割大量的水。 新树叶应在大约一周后从顶层节点开始生长。

    食谱

    1. NYGA(1升)
      5 g蛋白胨
      3g酵母提取物
      20ml 100%甘油
      1.5克琼脂
      1. 结合蛋白胨,酵母提取物和甘油,然后用水调至1 L,低热混合
      2. 用1M NaOH将pH调至7.0
      3. 在琼脂中混合。
      4. 高压灭菌30分钟。

    致谢

    这项工作由NSF/BREAD(资助0965418,BJS),NSF研究生研究奖学金(MC,MS)和NIH遗传学培训资助2T32GM007127-36A1(MC)资助。 pLAFR- lux 载体由Sebastian Schornack通过Frank Thieme提供。感谢Rose Kantor帮助MS接种图1所示的接种物。中期生长测定方案改编自Castiblanco等人(2013)。

    参考文献

    1. Castiblanco,LF,Gil,J.,Rojas,A.,Osorio,D.,Gutierrez,S.,Munoz-Bodnar,A.,Perez-Quintero,AL,Koebnik,R.,Szurek,B.,Lopez,C 。,Restrepo,S.,Verdier,V。和Bernal,AJ(2013)。 TALE1从 Xanthomonas axonopodis pv。 在植物细胞中作为转录激活子,并且对木薯植物的致病性是重要的。 Mol Plant Pathol 14(1):84-95。 />
    2. Cohn,M.,Bart,R.S.,Shybut,M.,Dahlbeck,D.,Gomez,M.,Morbitzer,R.,Hou,B.H.,Frommer,W​​.B.,Lahaye,T.and Staskawicz, Xanthomonas axonopodis 毒力是由转录激活子样效应物介导的在木薯中诱导SWEET糖转运蛋白。 Mol Plant Microbe Interact 27(11):1186-1198。
    3. Do Amaral,A.M.,Toledo,C.P.,Baptista,J.C.and Machado,M.A。(2005)。 转型 Xanthomonas axonopodis pv。 。 30:292-294。
    4. Howeler,R.,Lutaladio,N.和Thomas,G。(2013)。 节约与增长:Cassava:可持续生产集约化指南 食品和农业 联合国组织。
    5. López,C. E.和Bernal,A. J.(2012年)。 木薯细菌性枯萎病:使用基因组学来解释和管理一个老问题。/a> Trop Plant Biol 5(1):117-126。
    6. Lozano,J.C。(1986)。 木薯细菌性枯萎病:一种可控的疾病。 Plant Dis 70:1089-1093。
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    引用:Cohn, M., Shybut, M., Dahlbeck, D. and Staskawicz, B. (2015). Assays to Assess Virulence of Xanthomonas axonopodis pv. manihotis on Cassava . Bio-protocol 5(13): e1522. DOI: 10.21769/BioProtoc.1522.
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