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Surface Inoculation and Quantification of Pseudomonas syringae Population in the Arabidopsis Leaf Apoplast
拟南芥叶质外体中丁香假单胞菌种群的表面接种和定量   

Cristián  JacobCristián Jacob* Shweta  PanchalShweta Panchal* Maeli  MelottoMaeli Melotto  (*contributed equally to this work)
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Abstract

Bacterial pathogens must enter the plant tissue in order to cause a successful infection. Foliar bacterial pathogens that are not able to directly penetrate the plant epidermis rely on wounds or natural openings to internalize leaves. This protocol describes a procedure to estimate the population size of Pseudomonas syringae in the leaf apoplast after surface inoculation of Arabidopsis rosettes.

Keywords: Leaf inoculation(叶接种), Stomatal defense(气孔防御), Pseudomonas syringae(丁香假单胞菌), Foliar internalization(叶片内化), Apoplastic bacterial population(质外体细菌种群)

Background

Plant pathogenic bacteria causing foliar diseases may penetrate the leaf epidermis through wounds and natural openings such as stomata. Stomata are microscopic pores that mediate the regulation of transpiration and the exchange of gases between the plant and the atmosphere. Interestingly, we have demonstrated that bacteria can induce stomatal closure. This phenomenon is now recognized as stomatal defense, which hampers bacterial internalization into the leaf decreasing disease development (reviewed by Melotto et al., 2017, in press). Here, we describe a method adapted from Katagiri et al. (2002) and Panchal et al. (2016a and 2016b) to measure the total endophytic bacterial population of Pseudomonas syringae within Arabidopsis leaf tissue after surface inoculation. This procedure is useful to estimate bacterial penetration of leaves through stomata in a laboratory setting.

Materials and Reagents

  1. 3.5-inch square pots with holes (Hummert International, catalog number: 12-1300-1 )
  2. Soil mix, SunGro Sunshine® #1 Mix (Crop Production Services, catalog number: 1000590701 ) or equivalent
  3. Fine vermiculite (Growers Solution, catalog number: Vermiculite4cf )
  4. 48 inch x 100 ft Charcoal fiberglass screen (The Home Depot, catalog number: 3000016 )
  5. 15 ml and 50 ml centrifuge tubes
  6. Plastic domes (Hummert International, catalog number: 65-6964-1 )
  7. Sharpie markers, paper towels, Kimwipes, disposable gloves, rubber bands, forceps
  8. Square Petri dishes (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 240835 )
  9. Micropipettes (Rainin Pipet-LiteTM)
  10. 1.5 ml microfuge tubes
  11. Plastic pestles that fit 1.5 ml microfuge tubes (SP Scienceware - Bel-Art Products - H-B Instrument, catalog number: 19923-0001 )
  12. Plastic trays without holes (Hummert International, catalog number: 65-6963-1 )
  13. Microbreathe face mask (VWR, catalog number: 10833-224 )
  14. Arabidopsis thaliana (L. Heyhn.) ecotype Columbia (Col-0, ABRC stock CS60000). Seeds can be stored at 4 °C and are viable for 3-4 years
  15. Pseudomonas syringae bacterial culture (stored in 25% glycerol at -80 °C)
  16. Gnatrol (Hummert International, catalog number: 01-2035-1 )
  17. Arabidopsis controlled release fertilizer (LEHLE SEEDS, catalog number: PM-11 )
  18. Appropriate antibiotic (e.g., Rifampicin)
  19. MgCl2 solution
  20. Glycerol (MP Biomedicals, catalog number: 151194 )
  21. Silwet L-77 (LEHLE SEEDS, catalog number: VIS-30 )
  22. Reagent alcohol (Sigma-Aldrich, catalog number: 793183 )
  23. Sterile distilled water
  24. Agarose (VWR, catalog number: 97062-250 )
  25. Tryptone (IBI Scientific, catalog number: 41116105 )
  26. Yeast extract (U.S.Biotech Sources, catalog number: Y01PD-500 )
  27. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-500 )
  28. Bacteriological agar (IBI Scientific, catalog number: IB49171 )
  29. 0.1% agarose (see Recipes)
  30. Low-sodium Luria Bertani medium (see Recipes)

Equipment

  1. 1 ml micropipette
  2. Refrigerator or a cold room
  3. Plant growth chamber (Caron Products & Services, model: 6341-2 )
  4. Shaker incubator (VWR, catalog number: 12620-946 )
  5. Spectrophotometer (Thermo Fisher Scientific, model: Spectronic 20D+ or equivalent)
  6. Centrifuge (Eppendorf, model: 5810 )
  7. Handheld electric drill (BLACK + DECKER, catalog number: LDX120C )
  8. Cork borer No. 2 (Cole-Parmer, catalog number: EW-06298-98 )
  9. Digital hygrometer (VWR, catalog number: 35519-047 )
  10. Quantum meter (Apogee, catalog number: BQM )
  11. Vortex (BioExpress, GeneMate, catalog number: S-3200-1 )
  12. Autoclave
  13. laminar flow hood

Procedure

  1. Growing Arabidopsis plants
    1. Fill pots with the soil mix and top it off with a 2-cm layer of vermiculite (do not pack too tightly). Cover the pot with 7 x 7-inch fiberglass mesh screen (which prevents the leaves from contacting soil) and hold it in place with a rubber band.
    2. Place the pots in a tray and soak soil with an aqueous solution of Gnatrol (1 g L-1). Let it sit for one day.
      Note: This step is used to avoid development of fungus gnats.
    3. Mix Arabidopsis seeds in 5-10 ml of 0.1% agarose in a 15-ml centrifuge tube. Vortex the suspension for 10 sec at maximum speed to dispense the seeds evenly in agarose prior to sowing.
    4. Using a 1 ml micropipette, spot seeds (~5 seeds in one spot) in the four corners and the center of the pot. Label each pot with name, date, and genotype of seeds.
    5. Cover the tray with a plastic dome, leaving an opening of about 5-10 cm. Keep the tray in a cold room (4-8 °C) for 2 days to allow for efficient and synchronous germination (Figure 1A).


      Figure 1. Dip-inoculation procedure. A. Arabidopsis seeds are sown on a mesh-covered pot and kept in a cold room (4-8 °C) for 2 days to allow for efficient and synchronous germination. B. Arabidopsis 4-5 weeks old plants are used for inoculation. C. The rosette is dipped and gently swirled in the inoculum. D. Inoculated plants are placed in a tray and incubated in an environment chamber.
      Note: If a growth chamber without relative humidity control is used, cover the tray with a plastic dome leaving a 5-10 cm gap open to keep air relative humidity between 60-70%.

    6. After two days, place the tray in growth chamber set at 22 °C, 65 ± 5% relative humidity (RH), and a 12-h photoperiod under photosynthetic active light intensity of 100 µmol m-2 sec-1.
    7. When seedlings are at the 2-leaf stage, thin the spots with forceps such that only one seedling is left at each spot and all 5 plants in the pot look about the same age. Add 0.2 g fertilizer per liter of soil mix as suggested by the manufacturer. Water is added to the tray as needed; generally, 1 L of water per tray 2 or 3 times per week is sufficient. Do not overwater to avoid moisture buildup, growth of algae, and fungus gnat proliferation.

  2. Bacterial inoculum preparation
    1. From the glycerol stock, streak Pseudomonas syringae from a culture stock on low-salt Luria Bertani (LS-LB) medium supplemented with appropriate antibiotic (e.g., Pseudomonas syringae pv. tomato strain DC3000 is cultured in media containing 100 µg ml-1 of rifampicin). Incubate the plates at 28 °C for approximately 30 h. Once colonies appear on the plate, it can be stored at 4 °C for up to 10 days.
    2. In the morning of the day before inoculation, prepare the pre-inoculum by inoculating one colony from this fresh plate into 10 ml of LS-LB with appropriate antibiotic. Additionally, incubate 10 ml of LS-LB to use as a control for culture contamination and to set the standard (blank) for the spectrophotometer reading. Incubate the culture tubes at 28 °C and 200 rpm.
    3. After growing the pre-inoculum for approximately 8 h take the optical density (OD) at 600 nm using a spectrophotometer. To make the inoculum, dilute the pre-inoculum to 300 ml of fresh liquid LS-LB medium and the appropriate antibiotic to obtain an OD600 of 0.004. Use the equation V1 x C1 = V2 x C2. A 10-ml blank should also be included to calibrate the spectrophotometer.
    4. Incubate at 28 °C and 200 rpm until mid to late log phase is reached (OD600 of 0.8 to 1). It will take approximately 12 h.
    5. Collect the bacterial cells by centrifugation at 2,600 x g for 20 min at room temperature.
    6. Resuspend the cell pellet in sterile, distilled water to an OD600 of 0.2, which corresponds to the final inoculum concentration of 1 x 108 CFU ml-1. High inoculum concentration is required to ensure uniform infection of the leaf and avoid sampling problems and large experimental errors. Inoculum must be used immediately to avoid killing bacterial cells. Alternatively, a 1-10 mM MgCl2 solution can be used to resuspend bacterial cells and minimize possible damage associated with osmotic pressure. 
    7. To accurately estimate the inoculum concentration, prepare a 10-time serial dilution of the inoculum and plate each dilution on square plates. See the description of serial dilution procedure in the section below (enumeration of apoplastic bacterium population).
    8. Add 0.02% Silwet L-77 to the inoculum to ensure that leaves are evenly covered with the inoculum; otherwise, inoculum will runoff and infection will be spotty. This step is required to guarantee reproducibility of the results.

  3. Leaf surface inoculation by dipping
    1. Use 4-5-week-old Arabidopsis plants with fully expanded leaves for infection (Figure 1B).
    2. Acclimate plants at 25 °C for approximately 12 h before inoculation. Environmental condition, other than temperature, must be the same as the used for plant growth.
    3. Dip-inoculate plants as shown in Figure 1C and Video 1. Swirl leaves for 5 sec to make sure all leaves are evenly coated with the inoculum. Drip excess inoculum and place the pot back in the tray (Figure 1D).

      Video 1. Dip inoculation of Arabidopsis plants

    4. Add some water in the tray and cover the tray leaving around 5-10 cm gap open such that air relative humidity of 60-65% is maintained.
      Note: High relative humidity (> 70%) is not recommended as it may interfere with stomatal defense (Panchal et al., 2016a). It is important to inoculate plants at the same time of the day to ensure reproducibility across biological replicates. It is well documented that the plant immune response and stomatal movement vary with the circadian rhythm (Zhang et al., 2013).
    5. Place the trays in an environmental chamber set at 25 °C, 60-65% RH, and a 12-h photoperiod under photosynthetic active light intensity of 100 µmol m-2 sec-1. If the environmental chamber has a humidity control, the plastic dome is not needed.

  4. Enumeration of apoplastic bacterium population
    1. Pluck three leaves from a single plant at the petiole making sure the leaf blade is not damaged by the forceps tip. Fully expanded leaves from the second layer from rosette bottom can be used (see healthy looking plants in Figure 2A).
      Note: It is highly recommended to do a time course experiment up to 3 days after inoculation. Grow all plants in the same batch for each experiment and use different plants from the same batch for each time point. The time course must include sampling right after inoculation (day 0) to obtain the initial level of total live bacterial population on the plant. For this first time point, leaves should not be sterilized as described in step D2. Do not use the younger (top) or older (bottom) leaves of the rosette, or stressed leaves with curved edges or purpling. For description of Arabidopsis growth stages, please see Boyes et al. (2001).


      Figure 2. Enumeration of the bacterial population in the leaf. A. Three equal sized infected leaves are plucked and surface sterilized with 70% ethanol. B. Fours leaf disks are cut from each leaf using a cork borer. C and D. Four leaf disks diluted and 10 μl from each dilution is spotted twice (technical replicates) on square plates. Repeat B-D for each of the three leaves.

    2. Place the leaves in 70% ethanol for 2 min. Next, put the leaves in sterile water for 1 min while rinsing it lightly from time to time in a way that the leaf blade is not damaged.
    3. Dry the leaves by blotting on paper towel.
    4. Sterilize the cork borer and forceps at every step from here on by rinsing in 70% ethanol and then in sterile water. Remove water from the cork borer by blotting on the paper towel.
    5. Using the cork borer number 2, which has an area of 0.125 cm2, punch out four leaf discs (total area of 0.5 cm2), two on either side of the midrib (Figure 2B).
    6. Use surface sterilized forceps to collect the punched-out leaf discs and put them in 100 µl sterile water in a microfuge tube (represented by the first tube in Figure 2C). Grind the tissue sample using a plastic pestle attached to a hand-held electric drill. No broken leaf pieces should be visible (see Video 2).

      Video 2. Leaf tissue sampling and homogenization

    7. Take 10 µl of this first solution and add to 90 µl of sterile water in another microfuge tube making a 1:10 dilution. Similarly, serially dilute until 10-6 for day 0 (Figure 2C) or until 10-8 for subsequent sampling time points.
    8. Pipet 10 µl from each tube and spot in one square of a Petri dish containing LS-LB medium with appropriate antibiotic (see Figure 2D). Each dilution should be spotted twice as technical replicates (first and second row on the plate shown in Figure 2D). (see Video 3)
    9. Repeat steps D5 to D8 (Figures 2B-2D) for each of the three leaves (Figure 2A).
    10. After plating all dilutions, let the plate air dry and incubate it at 28 °C for approximately 30 h.

      Video 3. Bacterial enumeration using a serial dilution plating method

Data analysis

Count single colonies forming units (CFU) from one of the dilutions (e.g., the fourth or fifth column on the plate illustrated in Figure 2D). Choose the dilution that yields a 10-100 CFU range. Estimate the bacterial population by multiplying the CFU by the dilution factor. To express the value as CFU/cm2, multiply the total CFU count by 2 as the total area of four leaf discs (Figure 2B) is 0.5 cm2. For example, if you count 25 colonies in the dilution lane of 10-5 (5th column), then the bacterial population will be 25 x 105 x 2 = 5 x 106 CFU/cm2. For each sample, there should be three biological replicates (3 leaves; Figure 2A) with 2 technical replicates (2 spots on the medium for each dilution; Figure 2D). Statistical analysis should be done by calculating the average (n = 6) and standard error using Microsoft Excel or any other statistical analysis software. Significance of the difference between two samples can be obtained by performing the Student’s t-test. Additional biological replicates must be performed by repeating the whole experiment with other plants to assess the robustness of the analysis. For scientific rigor, this experimental procedure should be repeated three times and each time should yield similar bacterial growth trends. See examples of data graphs in Panchal et al. (2016a).

Notes

  1. Be careful not to wound the leaves while picking them with forceps, otherwise the leaves will be squishy and hard to punch holes. Also, wounding will potentially allow 70% ethanol to enter inside the leaf tissue, killing the internal bacterial population and thus giving skewed results.
  2. For reducing variability in biological replicates, it is extremely crucial that plants are not stressed while growing and during the infection period. Light, temperature, and relative humidity should be kept constant. Acclimatize plants for at least 12 h in the same growth chamber where infected plants are going to be placed.
  3. Increasing the number of leaves sampled can reduce standard error.
  4. Growing bacterial cultures beyond OD600 of 1.0 does not give ideal infection results.
  5. Always use bacterial pathogen from fresh plate streaked from glycerol stock. Multiple sub-culturing may lead to loss of virulence.

Recipes

  1. 0.1% agarose
    Dissolve 0.1 g agarose in 100 ml sterile distilled water by heating
    Keep swirling the solution intermittently while it cools
    Store and use at room temperature
  2. Low-sodium Luria Bertani medium
    10 g/L tryptone
    5 g/L yeast extract
    5 g/L NaCl
    2.5% agar (only for solid medium)
    Adjust pH to 7.0
    Autoclave medium at 15 psi, 120 °C for 15 min
    Allow medium to cool down to about 55 °C and add appropriate antibiotic if needed
    Pour medium into plates in a laminar flow hood
    Store plates in plastic bags at 4 °C to avoid medium dehydration

Acknowledgments

This work was supported by a grant from the US National Institute of Allergy and Infectious Disease (5R01AI068718) to Dr. Maeli Melotto.

References

  1. Boyes, D. C., Zayed, A. M., Ascenzi, R., McCaskill, A. J., Hoffman, N. E., Davis, K. R. and Görlach, J. (2001). Growth stage-based phenotypic analysis of Arabidopsis: A model for high throughput functional genomics in plants. Plant Cell 13: 1499-1510.
  2. Katagiri, F., Thilmony, R. and He, S. Y. (2002). The Arabidopsis thaliana-Pseudomonas syringae interaction. Arabidopsis Book 1: e0039.
  3. Melotto, M., Zhang, L., Oblessuc, P. R. and He, S. Y. (2017). Stomatal defense a decade later. Plant Physiol (in press).
  4. Panchal, S., Chitrakar, R., Thompson, B. K., Obulareddy, N., Roy, D., Hambright, W. S. and Melotto, M. (2016a). Regulation of stomatal defense by air relative humidity. Plant Physiol 172(3): 2021-2032.
  5. Panchal, S., Roy, D., Chitrakar, R., Price, L., Breitbach, Z. S., Armstrong, D. W. and Melotto, M. (2016b). Coronatine facilitates Pseudomonas syringae infection of Arabidopsis leaves at night. Front Plant Sci 7: 880.
  6. Zhang, C., Xie, Q., Anderson, R. G. Ng, G., Seitz, N. C., Peterson, T., McClung, C. R., McDowell, J. M., Kong, D., Kwak, J. M. and Lu, H. (2013). Crosstalk between the circadian clock and innate immunity in Arabidopsis. PLoS Pathogens 9(6): e1003370.

简介

细菌病原体必须进入植物组织才能使感染成功。不能直接穿透植物表皮的叶状细菌病原体依赖于伤口或自然开口使叶片内化。该方案描述了在表达接种拟南芥花环之后,在叶片质外体中估计结核菌的种群大小的程序。

背景 引起叶面疾病的植物病原菌可能通过伤口和天然开放(如气孔)穿透叶片表皮。气孔是介导调节蒸腾作用和植物与大气之间的气体交换的微观孔。有趣的是,我们已经证明细菌可以诱导气孔关闭。这种现象现在被认为是气孔防御,其阻碍细菌内化进入叶减少的疾病发展(由Melotto等人,2017年,在新闻中综述)。在这里,我们描述了从Katagiri等人改编的方法。 (2002)和Panchal等人。 (2016a和2016b)测量表面接种后拟南芥叶组织中的丁香假单胞菌的总内生细菌群体。该程序可用于在实验室环境中评估通过气孔的叶片细菌渗透。

关键字:叶接种, 气孔防御, 丁香假单胞菌, 叶片内化, 质外体细菌种群

材料和试剂

  1. 3.5英寸方孔,有孔(悍马国际,目录号:12-1300-1)
  2. 土壤混合,SunGro阳光®#1混合(作物生产服务,目录号:1000590701)或等同物
  3. 精细蛭石(种植者解决方案,目录号:Vermiculite4cf)
  4. 48英寸x 100英尺木炭玻璃纤维屏(Home Depot,目录号:3000016)
  5. 15 ml和50ml离心管
  6. 塑料圆顶(悍马国际,目录号:65-6964-1)
  7. Sharpie标记,纸巾,Kimwipes,一次性手套,橡皮筋,镊子
  8. 方形培养皿(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:240835)
  9. 微量移液器(Rainin Pipet-Lite TM
  10. 1.5 ml离心管
  11. 适合1.5 ml微量离心管的塑料杵(SP Scienceware - Bel-Art Products - H-B Instrument,目录号:19923-0001)
  12. 无孔塑料托盘(悍马国际,目录号:65-6963-1)
  13. 微口罩(VWR,目录号:10833-224)
  14. 拟南芥(L.Heyhn。)生态型Columbia(Col-0,ABRC stock CS60000)。种子可以在4°C储存,可以存活3-4年
  15. 细菌培养物(在-80℃下储存在25%甘油中)
  16. Gnatrol(Hummert International,目录号:01-2035-1)
  17. 拟南芥控释肥料(LEHLE SEEDS,目录号:PM-11)
  18. 适当的抗生素(例如,利福平)
  19. MgCl 2 解决方案
  20. 甘油(MP Biomedicals,目录号:151194)
  21. Silwet L-77(LEHLE SEEDS,目录号:VIS-30)
  22. 试剂酒精(Sigma-Aldrich,目录号:793183)
  23. 无菌蒸馏水
  24. 琼脂糖(VWR,目录号:97062-250)
  25. Tryptone(IBI Scientific,目录号:41116105)
  26. 酵母提取物(U.S.Biotech Sources,目录号:Y01PD-500)
  27. 氯化钠(NaCl)(Fisher Scientific,目录号:S271-500)
  28. 细菌琼脂(IBI Scientific,目录号:IB49171)
  29. 0.1%琼脂糖(见食谱)
  30. 低钠Luria Bertani培养基(见食谱)

设备

  1. 1 ml微量移液管
  2. 冰箱或冷藏室
  3. 植物生长室(Caron Products& Services,型号:6341-2)
  4. 振荡器孵化器(VWR,目录号:12620-946)
  5. 分光光度计(Thermo Fisher Scientific,型号:Spectronic 20D +或等效物)
  6. 离心机(Eppendorf,型号:5810)
  7. 手持式电钻(BLACK + DECKER,目录号:LDX120C)
  8. 软木钻头2号(Cole-Parmer,目录号:EW-06298-98)
  9. 数字湿度计(VWR,目录号:35519-047)
  10. 量子计(Apogee,目录号:BQM)
  11. Vortex(BioExpress,GeneMate,目录号:S-3200-1)
  12. 高压灭菌器
  13. 层流罩

程序

  1. 生长拟南芥植物
    1. 用泥土填充锅,并用2厘米的蛭石层(不要太紧)填满。用7 x 7英寸的玻璃纤维筛网(防止叶子接触土壤)覆盖锅,并用橡皮筋将其保持在适当的位置。
    2. 将锅放在托盘中,用Gnatrol水溶液(1 g L -1)浸泡土壤。让它坐一天。
      注意:此步骤用于避免发现真菌g。。
    3. 将拟南芥种子在5-10ml 0.1%琼脂糖中的15ml离心管中混合。将悬浮液以最大速度旋转10秒钟,以便播种前在琼脂糖中均匀分配种子。
    4. 使用1ml微量移液管,在四个角落和锅的中心点播种子(一个点5个种子)。标记每个锅的种子的名称,日期和基因型。
    5. 用塑料圆顶盖住托盘,留下约5-10厘米的开口。将托盘放在冷藏室(4-8°C)中2天,以实现高效同步发芽(图1A)。


      图1.浸种接种程序。将拟南芥种子播种在网眼覆盖的锅上,并保存在冷室(4-8℃)中2天以允许有效和同步的萌发。拟南芥使用4-5周龄的植物进行接种。 C.将玫瑰花被浸泡,并在接种物中轻轻旋转。 D.将接种的植物放置在托盘中并在环境室中温育。
      注意:如果使用没有相对湿度控制的生长室,用塑料圆顶覆盖托盘,留下5-10厘米的间隙打开,以保持空气相对湿度在60-70%之间。

    6. 两天后,将托盘置于22°C,65±5%相对湿度(RH)的生长室中,在光合作用光强度为100μmol/平方米的条件下放置12小时光周期。秒 -1
    7. 当幼苗处于2叶期时,用镊子稀释斑点,使得每个斑点只留下一个幼苗,并且锅中的所有5株植物看起来相同的年龄。根据制造商的建议,每公升土壤混合物添加0.2克肥料。根据需要将水加入托盘;通常每个托盘1升水2或3次就足够了。不要过水,避免水分积聚,藻类生长和真菌增殖。

  2. 细菌接种物准备
    1. 从补充有适当抗生素(例如,假单胞菌属)的低盐Luria Bertani(LS-LB)培养基上的培养物中,从甘油原料,连续的丁香假单胞菌(Strepomonas syringae)将菌株DC3000在含有100μg/ml利福平的培养基中培养,将番茄红素 pv。将板在28℃孵育约30小时。一旦菌落出现在板上,可以在4℃下储存长达10天。
    2. 在接种前一天的早晨,通过将来自该新鲜板的一个菌落接种到具有适当抗生素的10ml LS-LB中来制备预接种物。另外,孵育10ml LS-LB作为培养污染的对照,并设定分光光度计读数的标准(空白)。在28℃和200rpm培养培养管。
    3. 在接种后生长大约8小时后,用分光光度计测定600nm处的光密度(OD)。为了制备接种物,将预接种物稀释到300ml新鲜液体LS-LB培养基和合适的抗生素中以获得0.004的OD 600。使用方程式V 1 x C 1 = V 2 x C 2······还应包括10 ml空白以校准分光光度计。
    4. 在28℃和200rpm下孵育直至达到中间到晚期对数期(OD 600)为0.8〜1。大约需要12小时。
    5. 通过在室温下以2,600×g离心20分钟收集细菌细胞。
    6. 将细胞沉淀重新悬浮于无菌蒸馏水中至0.2的OD 600,其对应于1×10 8 CFU ml -1的最终接种物浓度,/sup>。需要高接种浓度以确保叶片的均匀感染,避免采样问题和大的实验误差。必须立即使用接种物以避免杀死细菌细胞。或者,可以使用1-10mM MgCl 2·2溶液来重悬细菌细胞并使与渗透压相关的可能的损害减到最小。
    7. 为了准确地估计接种物浓度,准备10次连续稀释接种物,并将每个稀释液平板放在方板上。请参阅以下部分中序列稀释程序的描述(排异型细菌群体的计数)。
    8. 将0.02%Silwet L-77加入到接种物中,以确保叶子均匀地被接种物覆盖;否则,接种物会流失,感染会很多。这一步是保证结果重现性所必需的。

  3. 通过浸渍叶面接种
    1. 使用具有完全扩增的叶片感染的4-5周龄的拟南芥植物(图1B)。
    2. 接种前在25°C适应植物约12 h。环境条件除温度以外,必须与用于植物生长的环境条件相同。
    3. 浸入接种植物,如图1C和视频1所示。旋转叶子5秒,以确保所有的叶子均匀地涂覆有接种物。滴入多余的接种物并将锅放回托盘(图1D)。

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      视频1.接种拟南芥植物
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    4. 在托盘中添加一些水,盖住托盘,约5-10厘米的间隙打开,保持空气相对湿度为60-65%。
      注意:不推荐高相对湿度(> 70%),因为它可能会干扰气孔防御(Panchal等,2016a)。重要的是在一天的同一时间接种植物以确保生物重复的重复性。据了解,植物免疫反应和气孔运动随着昼夜节律的不同而不同(Zhang et al。,2013)。
    5. 将托盘放在设定在25℃,60-65%RH的环境室中,并且在光合作用光强度为100μmol/平方米以上的条件下放置12小时光周期。如果环境室具有湿度控制,则不需要塑料圆顶。

  4. 排异型细菌群体的计数
    1. 从叶柄上的单一植物拔出三片叶子,确保叶片没有被镊子尖端损坏。可以使用来自莲座底部的第二层的完全膨胀的叶子(参见图2A中健康的植物)。
      注意:强烈建议在接种后3天内进行时间实验。为每个实验生长同一批次的所有植物,并在每个时间点使用同一批次的不同植物。时间过程必须包括在接种后(第0天)进行抽样,以获得植物上总活菌数量的初始水平。对于这个第一时间点,叶子不应该如步骤D2所述灭菌。不要使用玫瑰花的年轻(顶部)或较老的(底部)的叶子,或者应力叶子具有弯曲的边缘或紫色。有关拟南芥生长阶段的描述,请参见Boyes et al。 (2001)。


      图2.叶中细菌群体的计数。 A.采取三个相等大小的感染叶,并用70%乙醇表面灭菌。 B.使用软木钻孔机从每个叶子切割四叶片盘。将四个叶片稀释,每个稀释液10μl在方板上点样两次(技术重复)。对三个叶子中的每一个重复B-D。

    2. 将叶子放在70%乙醇中2分钟。接下来,将叶子置于无菌水中1分钟,同时不时地以叶片不损坏的方式漂洗。
    3. 用纸巾擦干叶子。
    4. 通过在70%乙醇中冲洗,然后在无菌水中,从这里每一步消毒软木蛀虫和镊子。通过在纸巾上吸墨去除软木蛀虫的水分。
    5. 使用面积为0.125厘米2的软木钻头2号,冲出四片圆盘(总面积为0.5厘米 2 ),两面在midrib(图2B)。
    6. 使用表面灭菌镊子收集冲出的叶盘,并将它们放入微量离心管(由图2C中的第一管表示)中的100μl无菌水中。使用附在手持电钻上的塑料杵研磨组织样本。没有破碎的叶片应该可见(见视频2)。

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      视频2.叶组织采样和均质
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    7. 取10μl第一溶液,加入另一个微量离心管中的90μl无菌水,稀释1:10。类似地,对于随后的取样时间点,对于第0天(图2C)或直到10 -8 连续稀释至10 -6。
    8. 从每个管中吸取10μl,并在含有具有适当抗生素的LS-LB培养基的培养皿的一平方的位点上点样(参见图2D)。每个稀释液应该是技术重复的两倍(图2D中的第一和第二排)。 (见视频3)
    9. 对于三个叶子中的每一个,重复步骤D5至D8(图2B-2D)(图2A)。
    10. 电镀所有稀释液后,让板空气干燥,并在28℃孵育约30小时。

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      视频3.使用串行稀释电镀方法进行细菌计数
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数据分析

从其中一个稀释液(例如,图2D中所示的板上的第四或第五列)计数单个菌落形成单位(CFU))。选择产生10-100 CFU范围的稀释度。通过将CFU乘以稀释因子来估计细菌群体。为了表示CFU/cm 2 的值,将总CFU计数乘以2,因为四个叶片的总面积(图2B)为0.5cm 2。例如,如果您计算10个浓度为10μL(5×sup)柱的稀释泳道中的25个菌落,则细菌群体将为25×10 5个//sup> x 2 = 5×10 <6>/CFU/cm 2。对于每个样品,应该有三个生物重复(3个叶子;图2A),具有2个技术重复(每个稀释的培养基上有2个点;图2D)。统计分析应通过使用Microsoft Excel或任何其他统计分析软件计算平均值(n = 6)和标准误差来进行。通过执行学生的测试可以获得两个样本之间的差异的显着性。必须通过与其他植物重复整个实验来评估其分析的鲁棒性来进行其他生物学重复。对于科学严谨性,该实验程序应重复三次,每次应产生类似的细菌生长趋势。参见Panchal等人中的数据图示例。 (2016a)。

笔记

  1. 小心不要用镊子捡伤叶子,否则叶子会很闷,难以打孔。此外,伤口将潜在地允许70%的乙醇进入叶组织内部,杀死内部细菌群体,从而产生倾斜的结果。
  2. 为了减少生物重复的变异性,植物在生长期间和感染期间都不会受到压力极其重要。光,温度和相对湿度应保持恒定。使植物在相同的生长室内适应环境至少12小时,在那里将放置受感染的植物。
  3. 增加采样叶数可以降低标准误
  4. 超过OD 600的生长细菌培养物不能产生理想的感染结果。
  5. 始终使用来自甘油原料条纹的新鲜板条的细菌病原体。多次亚培养可能导致毒力丧失。

食谱

  1. 0.1%琼脂糖
    通过加热将0.1g琼脂糖溶解在100ml无菌蒸馏水中 在冷却时不断地旋转溶液
    在室温下存放和使用
  2. 低钠Luria Bertani培养基
    10g/L胰蛋白胨
    5克/升酵母提取物
    5克/升NaCl
    2.5%琼脂(仅适用于固体培养基)
    调节pH至7.0
    高压灭菌介质在15psi,120℃下15分钟
    让培养基冷却至约55℃,如有需要可添加适当的抗生素 将介质倒入板状流层罩
    在4°C的塑料袋中放置板,以避免中度脱水

致谢

这项工作得到美国国家过敏和传染病研究所(5R01AI068718)授予Maeli Melotto博士的资助。

参考文献

  1. Boes,DC,Zayed,AM,Ascenzi,R.,McCaskill,AJ,Hoffman,NE,Davis,KR和Görlach,J。(2001)。拟南芥的基于生长阶段的表型分析:植物中高通量功能基因组学的模型植物细胞 13:1499-1510。
  2. Katagiri,F.,Thilmony,R.和He,SY(2002)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/22303207"target ="_ blank">拟南芥 - 丁香假单胞菌相互作用。拟南芥图书 1:e0039。
  3. Melotto,M.,Zhang,L.,Oblessuc,P.R。和He,S.Y。(2017)。气味防御十年后。植物生理学(在新闻中)。
  4. Panchal,S.,Chitrakar,R.,Thompson,BK,Obulareddy,N.,Roy,D.,Hambright,WS and Melotto,M.(2016a)。  通过空气相对湿度调节气孔防御。植物生理学172(3) :2021-2032。
  5. Panchal,S.,Roy,D.,Chitrakar,R.,Price,L.,Breitbach,ZS,Armstrong,DW和Melotto,M。(2016b)。< a class ="ke-insertfile"href = http://www.ncbi.nlm.nih.gov/pubmed/27446113"target ="_ blank"> Coronatine促进了晚期的拟南芥叶片的感染丁香假单胞菌。/a> 前植物科学 7:880.
  6. Zhang,C.,Xie,Q.,Anderson,RG Ng,G.,Seitz,NC,Peterson,T.,McClung,CR,McDowell,JM,Kong,D.,Kwak,JM和Lu,H。(2013 )  拟南芥中昼夜节律钟和先天免疫之间的串扰。病原体 9(6):e1003370。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Jacob, C., Panchal, S. and Melotto, M. (2017). Surface Inoculation and Quantification of Pseudomonas syringae Population in the Arabidopsis Leaf Apoplast. Bio-protocol 7(5): e2167. DOI: 10.21769/BioProtoc.2167.
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