参见作者原研究论文

本实验方案简略版
Apr 2021

本文章节


 

Bacterial Infection and Hypersensitive Response Assays in Arabidopsis-Pseudomonas syringae Pathosystem
拟南芥-丁香假单胞菌病理系统中的细菌感染和超敏反应测定   

引用 收藏 提问与回复 分享您的反馈 Cited by

Abstract

Arabidopsis thaliana-Pseudomonas syringae pathosystem has been used as an important model system for studying plant-microbe interactions, leading to many milestones and breakthroughs in the understanding of plant immune system and pathogenesis mechanisms. Bacterial infection and plant disease assessment are key experiments in the studies of plant-pathogen interactions. The hypersensitive response (HR), which is characterized by rapid cell death and tissue collapse after inoculation with a high dose of bacteria, is a hallmark response of plant effector-triggered immunity (ETI), one layer of plant immunity triggered by recognition of pathogen-derived effector proteins. Here, we present a detailed protocol for bacterial disease and hypersensitive response assays applicable to studies of Pseudomonas syringae interaction with various plant species such as Arabidopsis, Nicotiana benthamiana, and tomato.


Keywords: Arabidopsis thaliana (拟南芥), Pseudomonas syringae (丁香假单胞菌), Disease resistance (抗病能力), Hypersensitive response (超敏反应), Cell death (细胞死亡)

Background

Pseudomonas syringae is a Gram-negative phytopathogenic bacterial species that causes diseases on a broad host range, namely bacterial speck in tomato and canker disease in pepper and kiwifruit (Lewis Ivey and Miller, 2000; Basim et al., 2004; Mazzaglia et al., 2012; Xin and He, 2013). Over the last two decades, Pseudomonas syringae has also been an important model pathogen for studying bacterial ecology, pathogenesis mechanisms, and plant immune system (Xin et al., 2018). Due to its importance in basic biology research, as wells as in outbreaks of economically-important diseases, it was selected as the number one of the top 10 plant pathogenic bacteria in molecular plant pathology (Mansfield et al., 2012).


P. syringae bacteria are generally used as foliar pathogens in laboratories, although in nature they cause diseases in various organs. P. syringae enters plant leaf tissue through wounds or open stomata during natural infections, and uptakes nutrients in the apoplastic space of the leaves for multiplication (Xin and He, 2013). Bacterial disease assays are powerful tools in plant pathology studies. Two inoculation approaches, surface inoculation (i.e., by dipping or spray) and infiltration (i.e., by a needle-less syringe or vacuum), are commonly used in laboratories (Katagiri et al., 2002). Here we present step-by-step procedures for bacterial disease assays by syringe infiltration, which bypasses pathogen entry through stomata and plant “stomatal defense”, and is broadly used in studying plant “apoplast defense”. In addition, we also describe detailed procedures of the hypersensitive response assay, in which recognition of pathogen effectors by plant immune receptors triggers fast tissue cell death, and the rate of cell death can be used as a readout of the strength of plant immunity. Although this protocol is presented using the Arabidopsis-P. syringae pathosystem, it can be easily adapted to different pathosystems, such as Nicotiana benthamiana-P. syringae and tomato-P. syringae with slight modifications on bacterial inoculum.

Materials and Reagents

  1. Eppendorf tubes (1.5ml and 2 ml, Thermo Fisher Scientific, catalog number: 509-GRD-Q and 508-GRD-Q)

  2. 0.22 μm Millex-GP Syringe Filter (Merck, catalog number: SLGPR33RB)

  3. 96-well plate (200 μl, Round Bottom, Beyotime, catalog number: FPT016)

  4. Paper towels

  5. Pipette tips (Thermo Fisher Scientific QSP, catalog number:112NXL-Q)

  6. 1 ml needleless syringe (LABSTAR, catalog number: BX150)

  7. Arabidopsis thaliana accession Col-0, fec (Gimenez- Ibanez et al., 2009) and rps2 (Mindrinos et al., 1994)

    Note: Arabidopsis Col-0 plant contains the RPS2 gene, which mediates the recognition of effector protein AvrRpt2 and induces plant ETI resistance to Pst DC3000(avrRpt2). fls2 efr cerk1 (fec) triple mutant, which is mutated in three major pattern-recognition receptor genes; rps2 mutant is mutated in the RPS2 gene, encoding the receptor recognizing AvRpt2.

  8. Pseudomonas syringae pv. tomato (Pst) DC3000 and Pst DC3000(avrRpt2) (Mudgett et al., 1999)

  9. Sodium hypochlorite (Sinopharm Chemical Reagent, catalog number: 80010428)

  10. Sterilized water (e.g., Milli-Q)

  11. Mixed soil, which contains substrate (PINDSTRUP), vermiculite (Size: 1-3 mm) and perlite (Size: 3-5 mm), the ratio of these materials is 3:9:1 in mixed soil.

  12. Tryptone (OXOID, catalog number: LP0042B)

  13. Yeast Extract Powder (OXOID, catalog number: LP0021B)

  14. Potassium dihydrogen phosphate (KH2PO4) (Sinopharm Chemical Reagent, catalog number: 10017608)

  15. Sodium chloride (NaCl) (Sinopharm Chemical Reagent, catalog number: 10019318)

  16. Magnesium sulfate (MgSO4) (Sinopharm Chemical Reagent, catalog number: 20025117)

  17. Agar powder (Shanghai DingGuo Biotech, catalog number: DH010-1.1)

  18. Rifampicin (Yeasen Biotechnology, catalog number: 60234ES08)

  19. Spectinomycin (Sangon Biotech, catalog number: A600901-0005)

  20. 75% ethanol (Sinopharm Chemical Reagent, catalog number: 80176965)

  21. Luria-Marine (LM) solid medium (see Recipes)

  22. Rifampicin stock stock (50 g/L, 1,000×) (see Recipes)

  23. Spectinomycin stock stock (50 g/L, 1,000×) (see Recipes)

Equipment

  1. Ultra-low temperature freezer (-75°C freezer, New Brunswick Scientific)

  2. Tray (size: 310 g), transparent plastic dome, pot (size: 8 cm) and mesh (pore size: mesh 18 = 880 μm)

  3. Arabidopsis growth chamber (Percival and JIUPU)

  4. Pipette (1 ml, Rainin, model: L-1000PL for export)

  5. Centrifuge (Eppendorf, model: 5425R)

  6. Spectrophotometer (Thermo Fisher Scientific, model: NanoDrop ONEC)

  7. Steel ball (5 mm in diameter; SSCB, catalog number: KH000268)

  8. Millex-GP Syringe Filter Unit (Merck, catalog number: SLGPR33RB)

  9. Camera (Canon, model: EOS 80D)

  10. Vortex Oscillator (Scientific Industries, model: Vortex-Genie 2)

  11. Tweezer

  12. Beaker (Thermo Scientific, catalog number: 1201-1234)

  13. Cork borer (Sigma-Aldrich, catalog number: Z165220-1SET, 7.5 mm in diameter)

  14. TissueLyser (Shanghai Jingxin Industry, model: Tissuelyser-48)

  15. Stereoscope (Leica, model: MDG41)

  16. Autoclave

  17. Temperature and Humidity Data Logger (Easylog, model: EL-21CFR-2-LCD)

Software

  1. Microsoft Excel

  2. GraphPad Prism 8

Procedure

  1. Growing Arabidopsis plants in soil

    1. Sterilize Arabidopsis seeds with 5% sodium hypochlorite for 7-10 min, and then wash the seeds with sterilized water 5 times; place the sterilized seeds in the dark at 4°C for two days.

      Note: The cold treatment will synchronize germination.

    2. Place the mixed soil in an ultra-low temperature freezer (below -20°C) overnight.

      Note: Autoclaving soil is usually harmful for seed germination and plant growth in our hands. We therefore used a freezing treatment to kill insect eggs and larvae in the soil to prevent insect infestation during plant growth, without causing any visible effect on seed germination.

    3. Place the soil into small plastic pots, cover the pots with mesh, and fix the mesh with a rubber band (Figure 1A).

    4. Use a pipette to sow sterilized seeds (from A1, about 200-300 seeds/ml) in the soil (about 20-30 seeds per pot, 4-8 seeds at each corner).

    5. Grow plants in environmentally-controlled growth chambers, with relative humidity set at 60%, temperature at 22°C, light intensity at 100 μE⋅m-2⋅s-1 with a 12 h light/12 h dark photoperiod.

    6. Remove excess seedlings after one week, and keep 4 seedlings per pot.

    7. Water the plants with tap water every 2-3 days. Four- to five-week-old plants (Figure 1B) were used for our experiments.



      Figure 1. Experimental procedures of bacterial infection and hypersensitive response assays.

      A. Preparation of the mixed soil in pots. B. Appearance of 4-week-old Arabidopsis plants grown in environmentally-controlled growth chambers. C. Inoculation of leaves with bacterial solutions using a 1 ml needleless syringe. D. Inoculated plants are covered with a dome to keep high humidity, for the disease to develop in the greenhouse. E. Photograph of inoculated leaves 3 days after infiltration. F. Disease phenotype 3 days after infiltration in Col-0 plants, the white dotted box represents selected representative samples. G. Sterilization and rinsing of the sampled leaves. H. Sampling of leaf discs using a cork borer (7.5 mm in diameter). I. Ground leaf solution. J. Dilute the extracted solutions in different dilution ratios, and then take 10 μl from each dilution and place on LM agar plates. K. Count the colonies with a stereoscope. L. Photographs of tissue collapse phenotype for HR assay. Pst DC3000 (avrRpt2) bacteria were infiltrated at OD600 of 0.2 and images were taken about 7 h post infiltration (hpi).


  2. Preparing Pst strains for inoculation

    1. Streak out the Pst strains from -75°C freezer onto Luria-Marine (LM) solid medium containing antibiotics, and allow to grow in a 30°C incubator for 2 days.

    2. Culture the bacterial strains in 4-6 ml LM liquid medium supplemented with the appropriate antibiotics [50 mg/L rifampicin for Pst DC3000; 50 mg/L spectinomycin and 50 mg/L rifampicin for Pst DC3000(avrRpt2), which contains the pDSK600-avrRpt2 construct with spectinomycin resistance], shaking at 200 rpm and 30°C for 12-16 h.

      Note: The bacterial culture should reach mid-log growth phase (OD600 = 0.6-1.0).

    3. Transfer 1.5 ml bacterial culture to a 2 ml Eppendorf tube, and collect by centrifugation at 2,500 × g for 5 min.

    4. Remove the supernatant and resuspend the pellet with 2 ml sterilized water to wash.

    5. Centrifuge the bacterial solution at 2,500 × g for 5 min, and remove the supernatant, and then resuspend the pellet with 1 ml sterilized water.

    6. Adjust the bacterial solution to a cell density of OD600 = 0.2 (~1 × 108 cfu/ml) with sterilized water, measured with a spectrophotometer.


    For bacterial disease assay

    1. Inoculation of Arabidopsis with Pst strains

      1. Dilute the bacterial solution from B6 with sterilized water to a cell density from OD600 = 0.001 (~5 × 105 cfu/ml) to 0.002 (~1 × 106 cfu/ml).

      2. Inoculate 3 marked leaves (from the abaxial side of the leaves) per plant with adjusted bacterial solution using a 1 ml needleless syringe (Figure 1C and Video 1). We estimate that approximately 100-200 μl are necessary to fully infiltrate one adult leaf. We usually inoculate 4 plants for each strain, and inoculate different plants with different strains in each pot.


        Video 1. Syringe infiltration demonstration.


      3. Wipe off the solution on the surface of the infiltrated leaves with a paper towel.

      4. Keep inoculated plants under ambient humidity for about 1 h to allow evaporation of excess water from the leaf.

      5. Cover the tray with a transparent plastic dome to keep high humidity until sampling, and place plants back in the growth chamber for the disease to develop (Figure 1D).


    2. Recording the disease symptoms and counting the number of bacteria

      1. Harvest samples after 2-4 days (varies among experiments, due to different plant genotypes/bacterial strains), remove all inoculated leaves from the plants, and take a photo to record the chlorosis and necrosis symptoms (Figure 1E).

      2. Select 6-8 leaves that are representative of the symptoms (e.g., the middle level; Figure 1F), and place them in a 75% ethanol solution for ~30 s to kill the bacteria on the leaf surface.

      3. Place the leaves on the paper towel to quickly remove excess ethanol, and then rinse the leaves with sterilized water twice (Figure 1G).

      4. Dry the leaves with the paper towel, take two leaf discs from each leaf using a cork borer (7.5 mm in diameter) and four discs from two different leaves as one biological repeat, place the leaf discs into a 2 ml Eppendorf tube containing 200 μl of sterilized water and one or two steel balls (5 mm in diameter); collect three to four repeats from each treatment (Figure 1H).

      5. Grind the leaf discs by TissueLyser at 30 Hz, for 1 min.

      6. Quickly spin the extracted solutions (5,000 × g, 10 s) to move the solution from the tube caps to the inside of the tube; open the tube and add 800 μl of sterilized water to the tube, briefly vortex, and mix well by Vortex Oscillator (Figure 1I).

      7. Serially dilute the bacterial solutions with sterilized water (i.e., by 10×, 100×, 1,000×, etc.), and then take 10 μl from each dilution and place on LM agar plates supplemented with rifampicin (at 50 mg/L). Perform two technical replicates for each sample (Figure 1J), and air dry the plates at room temperature.

        Note: As 10 μl from 1 ml of the extracted solution were placed on a LM agar plate, this is the equivalent of a 100-fold dilution of the bacteria from 4 leaf discs. If we take 10 μl from 1 ml of the extracted solution to 90 μl of sterilized water and then take 10 μl to place on the LM agar plate, this is equivalent to another 10-fold dilution. Serial 10-fold dilutions are done for each sample by repeating this process. We usually dilute the extracted solution to 10-4, 10-5 and 10-6 for Pst DC3000, and dilute to 10-2, 10-3 and 10-4 for Pst DC3000 (avrRpt2).

      8. Place the air-dried LM agar plates in an incubator at 30°C for colonies to grow.

      9. Count the colonies with a stereoscope 24 h after incubation (Figure 1K). It can also be counted by eye if the colonies are well separated and grow to large sizes (e.g., after more than 24 h incubation).


      For the hypersensitive response assay

      1. Inoculation of Arabidopsis plants with Pst strains

        1. Inoculate 3-4 marked leaves (from the abaxial side of the leaves) per plant with Pst DC3000(avrRpt2) strain at a cell density of OD600 = 0.2 (~108 cfu/ml) using a 1 ml needleless syringe (Figure 1C). Inoculate 4-6 plants for each genotype.

        2. Wipe off the solution on the surface of the leaves with a paper towel, let the leaves dry, and place the plants back in growth conditions without cover and under ambient humidity. Check tissue collapse starting from 4-5 h after infiltration.

          Note: Different effector proteins lead to different ETI response intensities and different rates of HR, so different strains may require distinct observation time for HR.

        3. Harvest leaves, count the number of leaves showing cell death, and take a photo (Figure 1L).

          Note: When cell death occurs, ions will leak out from the cell, and the electrolyte leakage can be measured using a Electrolytic conductivity meter over a time course after infiltration, providing a more quantitative way of assessing HR (protocols described in Hatsugai and Katagiri, 2018).

      Data analysis

      Analysis of the data from disease assay:

      1. Enter the number of bacteria from dilutions (we usually choose the dilutions with 10-100 colonies for calculation) with corresponding dilution factor in Microsoft Excel.

      2. Average the two replicates of each sample. Multiply this average by a multitude of dilution factor to get the total number of colonies (colony forming units, CFU) in the sample. For example, if we get an average of 56 and the dilution factor is 10-5, then the total number of colonies is 56 × 105 CFU.

      3. Calculate the total leaf area. The area of one leaf disc of 7.5 mm diameter is equal to π multiplied by 0.375 squared, so the area is 0.441786 cm2; there are four leaf discs in one sample, so the total leaf area is 1.767 cm2.

      4. Divide the total number of bacteria of each sample by the leaf area to get the number of colonies per unit area. This value represents the disease susceptibility of the plant, and the higher the value, the more susceptible the plant is. For example, the number of colonies per unit area of the above sample from step1 is 56 × 105/1.767 = 3169213 CFU/cm2.

      5. Take the logarithm of base 10 of the number of colonies per unit area of each sample. For example, the above value from step 4 is Log10 3169213 = 6.5 Log10 (CFU/cm2).

      6. Enter all values from all samples in GraphPad, and calculate the average and standard deviation (SD) of 3 biological replicates, draw the figure and determine statistical significance using two-way ANOVA with Tukey’s test by GraphPad (Figure 2).



        Figure 2. Example graph of disease infection assay.

        The above graph was generated from one biological replicate of disease assay from Yuan et al. (2021) . Pst DC3000 (avrRpt2) bacteria were infiltrated into Arabidopsis leaves at OD600 of 0.002 and populations were determined at 3 dpi (mean ± S.D.; n = 3 biologically independent samples). Data were analyzed using two-way ANOVA with Tukey’s test.

      Notes

      1. We found that plant health status is one of the key determinants of disease phenotypes. Stressed or sub-optimal plants often give inconsistent or even opposite results. Abiotic conditions (such as light intensity/photoperiod, temperature, and water/soil wetness) in the chamber influence the health and basal defense level of plants. It’s important to use healthy plants that are grown under optimized conditions, and have a minimized or low level of basal defense for pathogen-related assays. We usually use 10 or 12 h light, keep the watering frequency/amount (not watering too much each time and avoid leaving standing water in the tray), and use plants at appropriate ages (usually around 4 weeks, as older plants sometimes tend to get anthocyanin accumulation and higher basal defense). Plants that look dark green or purple in the center of the rosettes are usually not used in our assays. Plants that are infected by insects or fungi also can not be used.

      2. Leaf age may affect hormonal level and plant immunity. Leaves of a similar age should be used in these assays. We usually use 3 leaves that are middle-aged and fully expanded from each plant. Bacterial dose and sampling time (e.g., day 2 instead of day 3 or 4) can be adjusted in experiments, depending on plant condition, and bacterial strains used.

      3. Environmental factors such as light, temperature and humidity affect disease development and severity level. We found disease development to be very sensitive to air humidity, so we usually cover the inoculated plants with a transparent plastic dome (fully covered) to keep relatively high air humidity inside (Figure 1D, above 90% relative humidity) for 3-4 days.

      4. For hypersensitive response assays, wounding and leaf-age seem to have a major effect on cell death rate. We observed that mechanical wounding during syringe infiltration dramatically accelerates tissue collapse. Thus, care should be taken during injection with the syringe. We also found that younger leaves are usually associated with faster cell death, so leaves at similar age should be chosen for a fair comparison, or to observe a less obvious HR phenotype.

      Recipes

      1. Luria-Marine (LM) medium

        Tryptone 10 g/L

        Yeast Extract Powder 6 g/L

        KH2PO4 1.5 g/L

        NaCl 0.6 g/L

        MgSO4 0.35 g/L

        Dissolve the ingredients in distilled water, adjust the pH to 7 with 10 N sodium hydroxide (NaOH) solution, and autoclave the solution.

        For LM solid medium, add 15 g/l agar powder in liquid medium before autoclaving.

      2. Rifampicin stock solution (1,000×)

        Dissolve 2.5 g of the rifampicin powder in 50 ml dimethyl sulfoxide (DMSO) to make a 50 g/l stock solution, mix well by vortex, aliquot the solution into 1.5 ml Eppendorf tubes, and store at -20°C.

      3. Spectinomycin stock solution (1,000×)

        Dissolve 2.5 g of the spectinomycin powder in 50 ml sterilized water to make 50 g/l stock solution, mix well by vortex, sterilize the solution by filtering through a 0.22 μm Millex-GP Syringe Filter, aliquot into 1.5 ml Eppendorf tubes, and store at -20°C.

      Acknowledgments

      This work was supported by the Chinese Academy of Sciences, Center for Excellence in Molecular Plant Sciences/Institute of Plant Physiology and Ecology, National Key Laboratory of Plant Molecular Genetics and Chinese Academy of Sciences Strategic Priority Research Program (Type-B; project number: XDB27040211). M.Y. was supported by National Postdoctoral Program for Innovative Talents (project number: BX2021313). We would like to thank Dr. Jun Fan at China Agricultural University for the advice on freezing treatment of soil for insect control.

      Competing interests

      The authors declare no competing interests.

      References

      1. Basim, H., Basim, E., Yilmaz, S., Dickstein, E. R. and Jones, J. B. (2004). An Outbreak of Bacterial Speck Caused by Pseudomonas syringae pv. tomato on Tomato Transplants Grown in Commercial Seedling Companies Located in the Western Mediterranean Region of Turkey. Plant Dis 88(9): 1050.
      2. Gimenez-Ibanez, S.,Ntoukakis, V. and Rathjen, J. P. (2009). The LysM receptor kinase CERK1 mediates bacterial perception in Arabidopsis.Plant Signal Behav 4(6): 539-41.
      3. Hatsugai, N. and Katagiri, F. (2018). Quantification of Plant Cell Death by Electrolyte Leakage Assay. Bio-protocol 8(5): e2758.
      4. Katagiri, F., Thilmony, R. and He, S. Y. (2002). The Arabidopsis thaliana-Pseudomonassyringae interaction. Arabidopsis Book 1: e0039.
      5. Lewis Ivey, M. L. and Miller, S. A. (2000). First Report of Bacterial Canker of Pepper in Ohio. Plant Dis 84(7): 810.
      6. Mansfield, J., Genin, S., Magori, S., Citovsky, V., Sriariyanum, M., Ronald, P., Dow, M., Verdier, V., Beer, S. V., Machado, M. A., et al. (2012). Top 10 plant pathogenic bacteria in molecular plant pathology. Mol Plant Pathol 13(6): 614-629.
      7. Mazzaglia, A., Studholme, D. J., Taratufolo, M. C., Cai, R., Almeida, N. F., Goodman, T., Guttman, D. S., Vinatzer, B. A. and Balestra, G. M. (2012). Pseudomonas syringae pv. actinidiae (PSA) isolates from recent bacterial canker of kiwifruit outbreaks belong to the same genetic lineage. PLoS One 7(5): e36518.
      8. Mindrinos, M., Katagiri, F., Yu, G. L. and Ausubel, F. M. (1994). The A. thaliana disease resistance gene RPS2 encodes a protein containing a nucleotide-binding site and leucine-rich repeats. Cell 78(6):1089-99.
      9. Mudgett, M. B. and Staskawicz, B. J.(1999). Characterization of the Pseudomonas syringae pv. tomato AvrRpt2 protein: demonstration of secretion and processing during bacterial pathogenesis. Mol Microbiol 32(5): 927-41.
      10. Xin, X. F. and He, S. Y. (2013). Pseudomonas syringae pv. tomato DC3000: a model pathogen for probing disease susceptibility and hormone signaling in plants. Annu Rev Phytopathol 51: 473-498.
      11. Xin, X. F., Kvitko, B. and He, S. Y. (2018). Pseudomonas syringae: what it takes to be a pathogen. Nat Rev Microbiol 16(5): 316-328.
      12. Yuan, M., Jiang, Z., Bi, G., Nomura, K., Liu, M., Wang, Y., Cai, B. and Zhou, J. M. (2021). Pattern-recognition receptors are required for NLR-mediated plant immunity. Natrue 592(7852): 105-109.


简介

[摘要]拟南芥拟南芥 -丁香假单胞菌病理系统已被用作研究植物-微生物相互作用的重要模型系统,在理解植物免疫系统和发病机制方面取得了许多里程碑和突破。细菌感染和植物病害评估是研究植物-病原体相互作用的关键实验。过敏反应 (HR) 的特点是接种高剂量细菌后细胞迅速死亡和组织塌陷,是植物效应触发免疫 (ETI) 的标志性反应,是由识别病原体触发的一层植物免疫-衍生的效应蛋白。在这里,我们提出了适用于丁香假单胞菌研究的细菌疾病和过敏反应测定的详细方案与各种植物物种的相互作用,如拟南芥、本氏烟草和番茄。

[背景] 丁香假单胞菌是一种革兰氏阴性植物病原菌,可在广泛的宿主范围内引起疾病,即番茄中的细菌斑点和辣椒和猕猴桃中的溃疡病(Lewis Ivey 和 Miller,2000;Basim等,2004;Mazzaglia等,2012;Xin 和 He,2013)。在过去的二十年里,丁香假单胞菌也是研究细菌生态学、发病机制和植物免疫系统的重要模式病原体(Xin et al. , 2018)。由于其在基础生物学研究以及经济上重要疾病的爆发中的重要性,它被选为分子植物病理学中排名前 10 位的植物病原菌之一(Mansfield等,2012)。
丁香假单胞菌细菌通常在实验室中用作叶面病原体,尽管它们在自然界中会引起各种器官的疾病。丁香假单胞菌在自然感染过程中通过伤口或开放的气孔进入植物叶片组织,并在叶片的质外空间吸收养分进行繁殖(Xin and He,2013)。细菌疾病检测是植物病理学研究中的有力工具。实验室通常使用两种接种方法,即表面接种(即通过浸渍或喷雾)和浸润(即通过无针注射器或真空)(Katagiri等,2002)。在这里,我们介绍了通过注射器渗透进行细菌疾病检测的分步程序,它绕过了病原体通过气孔和植物“气孔防御”进入,并广泛用于研究植物“质外体防御”。此外,我们还描述了过敏反应测定的详细程序,其中植物免疫受体识别病原体效应物触发快速组织细胞死亡,细胞死亡率可用作植物免疫强度的读数。尽管使用所呈现的该协议拟南芥-丁香假单胞菌病害系统,它可以很容易地适应于不同的pathosystems,如本塞姆氏烟草-丁香假单胞菌和tomato-丁香假单胞菌与细菌接种物轻微的修改。

关键字:拟南芥, 丁香假单胞菌, 抗病能力, 超敏反应, 细胞死亡

材料和试剂

 

1.     Eppendorf 管(1.5ml 2mlThermo Fisher Scientific,目录号:509-GRD-Q 508-GRD-Q

2.     0.22 μm Millex-GP 注射器过滤器(默克,目录号:SLGPR33RB

3.     96 孔板(200 μlRound BottomBeyotime,目录号:FPT016

4.     纸巾

5.     移液器吸头(Thermo Fisher Scientific QSP,目录号:112NXL-Q

6.     1 ml无针注射器(LABSTAR,目录号:BX150

7.     拟南芥种质 Col-0fec Gimenez-Ibanez2009)和rps2 Mindrinos1994

注:拟南芥 Col-0 植物含有 RPS2 基因,该基因介导效应蛋白 AvrRpt2 的识别并诱导植物 ETI Pst DC3000(avrRpt2) 的抗性。fls2 efr cerk1 fec )三重突变体,在三个主要模式识别受体基因中发生突变;rps2 突变体在 RPS2 基因中发生突变,编码识别 AvRpt2 的受体。

8.     丁香假单胞菌光伏。番茄 (Pst) DC3000 Pst DC3000( avrRpt2 )Mudgett1999

9.     次氯酸钠(国药化学试剂,目录号:80010428

10.  无菌水(例如Milli-Q

11.  混合土,包含基质(PINDSTRUP)、蛭石(尺寸:1-3 mm)和珍珠岩(尺寸:3-5 mm),这些材料在混合土壤中的比例为3:9:1

12.  胰蛋白胨(OXOID,目录号:LP0042B

13.  酵母提取物粉(OXOID,目录号:LP0021B

14.  磷酸二氢钾(KH PO )(国药化学试剂,目录号:10017608

15.  氯化钠(NaCl)(国药集团化学试剂,目录号:10019318

16.  硫酸镁(MgSO )(国药集团化学试剂,目录号:20025117

17.  琼脂粉(上海鼎国生物,目录号:DH010-1.1

18.  利福平(Yeasen Biotechnology,目录号:60234ES08

19.  壮观霉素(Sangon Biotech,目录号:A600901-0005

20.  75%乙醇(国药化学试剂,目录号:80176965

21.  Luria-Marine (LM) 固体培养基(见配方)

22.  利福平原液(50 g/L1,000×)(见配方)

23.  壮观霉素原液(50 g/L1,000×)(见配方)

 

设备

 

1.     超低温冷冻机(-75°C冷冻机New Brunswick Scientific

2.     托盘(尺寸:310 g)、透明塑料圆顶、锅(尺寸:8 cm)和网(孔径:网 18 = 880 μm

3.    拟南芥生长室(Percival JIUPU

4.     移液器(1 mlRainin,型号:L-1000PL 出口)

5.     离心机(Eppendorf,型号:5425R 

6.     分光光度计Thermo Fisher Scientific型号:NanoDrop ONE 

7.     钢球(直径 5 mmSSCB,目录号:KH000268              

8.     Millex-GP 注射器过滤器单元(默克,目录号:SLGPR33RB

9.     相机(佳能,型号:EOS 80D

10.  Vortex Oscillator(科学工业,型号:Vortex-Genie 2

11. 

12.  烧杯(Thermo Scientific目录号:1201-1234              

13.  软木钻孔器(Sigma-Aldrich目录号: Z165220-1SET,直径7.5 mm

14.  TissueLyser(上海精信实业,型号:Tissuelyser-48

15.  立体镜(徕卡,型号:MDG41

16.  高压釜

17.  温湿度数据记录器(Easylog,型号:EL-21CFR-2-LCD

 

软件

 

1.     微软Excel

2.     GraphPad棱镜8

 

程序

 

A.     在土壤中种植拟南芥植物

1.     拟南芥种子用5%次氯酸钠灭菌7-10分钟,然后用灭菌水冲洗种子5次;将消毒后的种子°C的黑暗中放置两天。

注意:冷处理会同步发芽。

2.     将混合好的土壤放入超低温冰箱(-20°C 以下)中过夜。

注意:高压灭菌土壤通常对我们手中的种子发芽和植物生长有害。因此,我们使用冷冻处理来杀死土壤中的昆虫卵和幼虫,以防止植物生长过程中的昆虫侵扰,而不会对种子萌发造成任何明显影响。

3.     土壤放入小塑料盆中,用网盖住盆,并用橡皮筋固定网(图 1A)。

4.     用移液管在土壤中播种消毒种子(来自 A1,约 200-300 粒种子/ml)(每盆约 20-30 粒种子,每个角 4-8 粒种子)。

5.     60%,温度生长的植物在环境受控生长室,与相对湿度设定在22,在100μE光强度-2 小号-1具有12小时光照/ 12小时黑暗的光周期。

6.     一周后去除多余的幼苗,每盆保留4个幼苗。

7.     2-3 天用自来水给植物浇水。我们的实验使用了四到五周大的植物(图 1B

 

1. 细菌感染和过敏反应检测的实验程序。

A. 在盆中准备混合土壤。B.在环境控制的生长室中生长的 4 周龄拟南芥植物的外观C. 使用 1 ml 无针注射器用细菌溶液接种叶子。D. 接种的植物用圆顶盖住以保持高湿度,以便病害在温室中发展。E. 浸润后 3 天的接种叶照片。F.渗入 Col-0 植物 3 天后的疾病表型,白色虚线框代表选定的代表性样品。G. 对采样的叶子进行消毒和冲洗。H.使用软木钻孔器(直径 7.5 毫米)对叶盘进行采样。I. 地叶溶液。J. 将提取液以不同的稀释比例稀释,然后从每个稀释液中取 10 μl 置于 LM 琼脂平板上。K. 用立体镜计算菌落数。L. 用于 HR 测定的组织塌陷表型的照片。Pst DC3000 ( avrRpt2 ) 细菌在 OD 600 0.2 时浸润,并在浸润后 (hpi) 7 小时拍摄图像。

 

B.     准备接种Pst菌株

1.     条纹Pst-75冷冻机菌株上的Luria-海洋(LM)含有固体培养基的抗生素,和允许在一个30到生长孵化2天。

2.     在补充有适当抗生素的 4-6 ml LM 液体培养基中培养细菌菌株Pst DC300050 mg/L 利福平;50 mg/L 壮观霉素和 50 mg/L 利福平用于Pst DC3000 ( avrRpt2 ),其中包含具有壮观霉素抗性的pDSK600-avrRpt2构建体],在 200 rpm 30°C 下振荡 12-16 小时。

注意:细菌培养应达到对数中期(OD 600 = 0.6-1.0)。

3.     1.5 ml 细菌培养物转移到 2 ml Eppendorf 管中,以 2,500 × g离心5 分钟收集。

4.     去除上清液,用 2 ml 无菌水重悬沉淀以洗涤。

5.     将菌液以 2,500 × g离心5 分钟,除去上清液,然后用 1 ml 无菌水重悬沉淀。

6.     用无菌水将细菌溶液调整到 OD 600 = 0.2 (~1 × 10 cfu/ml)的细胞密度,用分光光度计测量。 

 

用于细菌疾病检测

C.    Pst菌株接种拟南芥

1.     用无菌水将 B6 中的细菌溶液稀释到细胞密度从 OD 600 = 0.001 (~5 × 10 cfu/ml) 0.002 (~1 × 10 cfu/ml)

2.     使用 1 ml 无针注射器(图 1C 和视频 1用调整后的细菌溶液为每株植物接种 3 个标记的叶子(从叶子的背面)。我们估计大约需要 100-200 μl 才能完全渗入一片成年叶。我们通常每株接种4株植物,每盆接种不同株系的不同植株

 

 

视频 1. 注射器渗透演示。

 

3.     用纸巾擦去渗入叶子表面的溶液。

4.     将接种过的植物在环境湿度下保持约 1 小时,以蒸发叶子中多余的水分。

5.     用透明塑料圆顶盖住托盘,以保持高湿度,直到采样,并将植物放回生长室,以便疾病发展(图 1D)。

 

D.    记录疾病症状并计算细菌数量

1.     2-4 天后收获样品(因不同的植物基因型/细菌菌株而异,因实验而异),从植物中取出所有接种的叶子,并拍照记录萎黄和坏死症状(图 1E

2.     选择代表症状的 6-8 片叶子(例如,中间层;图 1F),并将它们放入 75% 乙醇溶液中约 30 秒,以杀死叶表面的细菌。

3.     将叶子放在纸巾上以快速去除多余的乙醇,然后用消毒水冲洗叶子两次(图 1G)。

4.     用纸巾擦干叶子,使用软木钻孔器(直径 7.5 毫米)从每片叶子上取两个叶盘,从两片不同的叶子上取四个盘作为一个生物重复,将叶盘放入含有 200 μl 2 ml Eppendorf 管中消毒水和一到两个钢球(直径 5 毫米);从每次处理中收集三到四次重复(图 1H

5.     通过 TissueLyser 30 Hz 下研磨叶盘 1 分钟。

6.     快速旋转提取的溶液 (5,000 × g , 10 s),将溶液从管盖移至管内;打开管子,加入800 μl的无菌水到管子中,短暂涡旋,涡旋振荡器混匀(图1I

7.     用无菌水连续稀释菌液(10×100×1,000×),然后从每次稀释液中取10 μl置于添加了利福平(50 mg/L)的LM琼脂平板上对每个样品进行两次技术复制(图 1J,并在室温下风干板。

注意:将 1 ml 提取液中的 10 μl 置于 LM 琼脂平板上,这相当于将 4 个叶盘中的细菌稀释 100 倍。如果从1ml提取液中取10μl加入90μl无菌水中,再取10μl置于LM琼脂平板上,相当于再稀释10倍。通过重复此过程,对每个样品进行连续 10 倍稀释。对于 Pst DC3000,我们通常将提取的溶液稀释到 10 -4 10 -5 10 -6 ,对 Pst DC3000 (avrRpt2)稀释到 10 -2 10 -3 10 -4 

8.     将风干的 LM 琼脂平板置于 30°C 的培养箱中进行菌落生长。

9.     孵化后 24 小时用立体镜计数菌落(图 1K。如果菌落分离良好并长到大尺寸(例如,孵育超过 24 小时后),也可以通过肉眼计数。

 

对于超敏反应测定

E.     Pst菌株接种拟南芥植物

1.     使用 1 ml 无针注射器在细胞密度为 OD 600 = 0.2 (~10 cfu/ml) 的情况下,用Pst DC3000 ( avrRpt2 ) 菌株接种 3-4 个标记的叶子(从叶子的背面)(图 1C )。每个基因型接种 4-6 株植物。

2.     用纸巾擦去叶子表面的溶液,让叶子变干,然后将植物放回生长条件下,无需覆盖和环境湿度。从浸润后 4-5 小时开始检查组织塌陷。

注意:不同的效应蛋白导致不同的 ETI 反应强度和不同的 HR 率,因此不同的菌株可能需要不同的 HR 观察时间。

3.     收获叶子,计算显示细胞死亡的叶子数量,然后拍照(图 1L)。

注意:当发生细胞死亡时,离子会从细胞中泄漏出来,并且可以在渗透后的一段时间内使用电解电导率计测量电解质泄漏,从而提供一种更定量的评估 HR 的方法(Hatsugai Katagiri 中描述的协议, 2018)。

 

数据分析

 

疾病检测数据分析:

1.     Microsoft Excel 中输入带有相应稀释因子的稀释度(我们通常选择 10-100 个菌落的稀释度进行计算)中的细菌数。

2.     平均每个样品的两个重复。将此平均值乘以多个稀释因子,以获得样本中的菌落总数(菌落形成单位,CFU)。例如,如果我们得到平均值为 56,稀释因子为 10 -5 ,则菌落总数为 56 × 10 CFU

3.     计算总叶面积。一个直径7.5mm的叶盘的面积等于π乘以0.375的平方,所以面积是0.441786 cm ;一个样品中有四个叶盘,因此总叶面积为 1.767 cm 

4.     将每个样品的细菌总数除以叶面积,得到单位面积的菌落数。该值代表植物的病害易感性,值越高,植物越易感病。例如,上述 step1 样本单位面积的菌落数为 56 × 10 /1.767 = 3169213 CFU/cm 

5.     取每个样品单位面积菌落数的以10为底的对数。例如,上述步骤 4 中的值为 Log 10 3169213 = 6.5 Log 10 (CFU/cm )

6.     GraphPad 中输入所有样本的所有值,并计算 3 个生物重复的平均值和标准偏差 (SD),绘制图形并使用 GraphPad Tukey 检验使用双向 ANOVA 确定统计显着性( 2

 

2. 疾病感染检测的示例图。

上图是由袁等人的疾病检测的一个生物学重复产生的。(2021)Pst DC3000 ( avrRpt2 ) 细菌在 OD 600 0.002 时渗入拟南芥叶中,并在 3 dpi 时确定种群(平均值 ± SD= 3 个生物独立样本)。使用带有 Tukey 检验的双向 ANOVA 分析数据。

 

笔记

 

1.     我们发现植物健康状况是疾病表型的关键决定因素之一。受压或次优植物通常会产生不一致甚至相反的结果。室内的非生物条件(例如光强度/光周期、温度和水/土壤湿度)会影响植物的健康和基础防御水平。重要的是使用在优化条件下生长的健康植物,并且对病原体相关的检测具有最小化或低水平的基础防御。我们通常使用 10 12 小时光照,保持浇水频率/数量(每次不要浇太多,避免在托盘中留下积水),并在适当的年龄(通常在 4 周左右,因为老植物有时倾向于获得花青素积累和更高的基础防御)。在我们的分析中通常不使用在玫瑰花结中心看起来是深绿色或紫色的植物。被昆虫或真菌感染的植物也不能使用。

2.     叶龄可能会影响荷尔蒙水平和植物免疫力。在这些测定中应使用相似年龄的叶子。我们通常使用 3 片中年且从每株植物中完全展开的叶子。细菌剂量和采样时间(例如,第 2 天而不是第 3 天或第 4 天)可以在实验中进行调整,这取决于植物条件和使用的细菌菌株。

3.     光、温度和湿度等环境因素会影响疾病的发展和严重程度。我们发现疾病的发展对空气湿度非常敏感,因此我们通常用透明塑料圆顶(完全覆盖)覆盖接种的植物,以保持内部相对较高的空气湿度(图 1D,相对湿度高于 90%3-4 .

4.     对于过敏反应测定,伤口和叶龄似乎对细胞死亡率有主要影响。我们观察到注射器浸润过程中的机械损伤会显着加速组织塌陷。因此,在用注射器注射时应小心。我们还发现,较年轻的叶子通常与更快的细胞死亡有关,因此应该选择年龄相近的叶子进行公平比较,或者观察不太明显的 HR 表型。

 

食谱

 

1.     Luria-Marine (LM) 培养基

胰蛋白胨 10 /

酵母提取物粉末 6 g/L

KH PO 1.5 /

氯化钠 0.6 /

硫酸镁0.35 /

将成分溶解在蒸馏水中,用 10 N 氢氧化钠 (NaOH) 溶液将 pH 值调节至 7,然后将溶液高压灭菌。

对于 LM 固体培养基,在高压灭菌前在液体培养基中加入 15 g/l 琼脂粉。

2.     利福平原液 (1,000×)

2.5 g 利福平粉末溶解在 50 ml 二甲基亚砜 (DMSO) 中,制成 50 g/l 储备溶液,涡旋混合均匀,将溶液分装到1.5 ml Eppendorf 管中,并在 -20°C 下储存。

3.     壮观霉素原液 (1,000×)

2.5 g 壮观霉素粉末溶解在 50 ml 无菌水中制成 50 g/l 储备液,涡旋混合均匀,通过0.22 μm Millex-GP 注射器过滤器过滤消毒溶液,分装到 1.5 ml Eppendorf 管中,并储存在-20 °C

 

致谢

 

这项工作得到了中国科学院、分子植物科学卓越中心/植物生理生态研究所、植物分子遗传学国家重点实验室和中国科学院战略重点研究计划(B类;项目编号: XDB27040211)。国家博士后创新人才计划资助项目(项目编号:BX2021313)。感谢中国农业大学范俊博士对土壤冷冻处理防治昆虫提出的建议。

 

利益争夺

 

作者声明没有竞争利益。

 

参考

1.     Basim, H.Basim, E.Yilmaz, S.Dickstein, ER Jones, JB (2004)丁香假单胞菌引起的细菌斑点的爆发。在位于土耳其西地中海地区的商业苗木公司种植的番茄移植物上的番茄。植物 Dis 88(9): 1050

2.     Gimenez-Ibanez, S.,Ntoukakis, V. Rathjen, JP (2009)LysM 受体激酶 CERK1 介导拟南芥中的细菌感知。植物信号行为4(6): 539-41

3.     Hatsugai, N. Katagiri, F. (2018)通过电解质泄漏测定法定量植物细胞死亡。生物协议8(5)e2758

4.     Katagiri, F.Thilmony, R. He, SY (2002)拟南芥-假单胞菌丁香互动。 拟南芥书1e0039

5.     Lewis Ivey, ML Miller, SA (2000)俄亥俄州辣椒细菌性溃疡病的第一份报告植物 Dis 84(7): 810

6.     Mansfield, J., Genin, S., Magori, S., Citovsky, V., Sriariyanum, M., Ronald, P., Dow, M., Verdier, V., Beer, SV, Machado, MA. (2012)分子植物病理学中的十大植物病原菌。Mol Plant Pathol 13(6): 614-629

7.     Mazzaglia, A., Studholme, DJ, Taratufolo, MC, Cai, R., Almeida, NF, Goodman, T., Guttman, DS, Vinatzer, BA Balestra, GM (2012)丁香假单胞菌光伏。从最近爆发的猕猴桃细菌溃疡病中分离出的猕猴桃(PSA) 属于同一遗传谱系 PLoS 7(5)e36518

8.     Mindrinos, M.Katagiri, F.Yu, GL Ausubel, FM (1994)拟南芥抗病基因RPS2编码含有核苷酸结合位点和富含亮氨酸的重复序列的蛋白。 细胞78(6):1089-99

9.     Mudgett, MB Staskawicz, BJ(1999)丁香假单胞菌pv 表征。番茄 AvrRpt2 蛋白:细菌发病过程中分泌和加工的证明。Mol Microbiol 32(5): 927-41

10.  Xin, XF He, SY (2013)丁香假单胞菌光伏。番茄 DC3000:一种用于探测植物疾病易感性和激素信号传导的模型病原体 Annu Rev Phytopathol 51473-498

11.  Xin, XF, Kvitko, B. He, SY (2018)丁香假单胞菌:成为病原体需要什么。 Nat Rev Microbiol 16(5): 316-328

12.  Yuan, M.Jiang, Z.Bi, G.Nomura, K.Liu, M.Wang, Y.Cai, B. Zhou, JM (2021)NLR 介导的植物免疫需要模式识别受体。 自然592(7852)105-109

登录/注册账号可免费阅读全文
  • English
  • 中文翻译
免责声明 × 为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。
Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用:Yuan, M. and Xin, X. F. (2021). Bacterial Infection and Hypersensitive Response Assays in Arabidopsis-Pseudomonas syringae Pathosystem. Bio-protocol 11(24): e4268. DOI: 10.21769/BioProtoc.4268.
提问与回复

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。

如果您对本实验方案有任何疑问/意见, 强烈建议您发布在此处。我们将邀请本文作者以及部分用户回答您的问题/意见。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片的形式来说明遇到的问题。