An Improved Bioassay to Study Arabidopsis Induced Systemic Resistance (ISR) Against Bacterial Pathogens and Insect Pests
一种的可用于检测拟南芥中细菌性病原体和害虫诱导的系统抗性的改进生物学方法   

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Molecular Plant Microbe Interactions
Jan 2019

 

Abstract

The plant immune system is essential for plants to perceive and defend against bacterial, fungal and insect pests and pathogens. Induced systemic resistance (ISR) is a systemic immune response that occurs upon root colonization by beneficial microbes. A well-studied model for ISR is the association of specific beneficial strains of Pseudomonas spp. with the reference plant Arabidopsis thaliana. Here, we describe a robust, increased throughput, bioassay to study ISR against the bacterial pathogen Pseudomonas cannabina pv. alisalensis (formerly called Pseudomonas syringae pv. maculicola) strain ES4326 and the herbivore Trichoplusia ni by inoculating Pseudomonas simiae strain WCS417 (formerly called Pseudomonas fluorescens WCS417) on Arabidopsis plants grown in Jiffy-7® peat pellets. While most commonly used for Pseudomonas-triggered ISR on Arabidopsis, this assay is effective for diverse rhizosphere bacterial strains, plant species, pathogens and herbivores.

Keywords: Induced Systemic Resistance (ISR) (诱导系统抗性), Pseudomonas simiae (猴假单胞菌), Arabidopsis thaliana (拟南芥), Pseudomonas syringae (丁香假单胞菌), Pseudomonas cannabina (大麻假单胞菌), Trichoplusia ni (粉纹夜蛾)

Background

After plant infection or colonization by pathogenic or commensal microbes, a systemic defense response can ensue involving immunological “memory” or “priming” (Parker, 2009; Fu and Dong, 2013; Martinez-Medina et al., 2016). Depending on the microbe and the associated plant organ, different systemic resistance programs are induced (Pieterse et al., 2014). When roots encounter specific commensal microbes, induced systemic resistance (ISR) in distal root and shoot tissue is observed (Pieterse et al., 1996; van Loon et al., 1998). Although the genetic and hormonal signaling mechanisms deployed during the ISR response are partially understood (Verhagen et al., 2004; Pieterse et al., 2014; Pangesti et al., 2016), many questions still remain unanswered, such as the mechanisms involved in root-microbiome communication or the identity of systemic signal(s) (Pieterse et al., 2014; Haney et al., 2018; Cecchini et al., 2019).

The inoculation of Arabidopsis thaliana roots with Pseudomonas simiae WCS417 has been used as a model system for studying ISR against bacteria, fungi and herbivore invaders (Pieterse et al., 1996; van Loon et al., 1998; Verhagen et al., 2004; Haney et al., 2015). Existing bioassays for P. simiae-Arabidopsis ISR make use of sterile sand-germinated Arabidopsis followed by seedling transplantation into a sand/soil mixture containing the rhizobacteria (Pieterse et al., 1996; Pozo et al., 2008; van Oosten et al., 2008). Building on this method, we have recently developed ISR bioassays against the bacterial pathogen Pseudomonas cannabina pv. alisalensis (formerly called Pseudomonas syringae pv. maculicola) strain ES4326 (Pma) and the herbivore Trichoplusia ni (T. ni) using Arabidopsis plants germinated and grown in Jiffy-7® peat pellets. This is an effective system to robustly trigger and quantify ISR, primarily because peat pellets have a constant volume and low fluorescent pseudomonad content (Haney et al., 2015 and 2018; Cecchini et al., 2019). Moreover, avoiding the transplantation of seedlings reduces the experimental labor and stress to the seedlings, thereby improving assay efficiency. Here, we describe a step-by-step bioassay methodology for ISR experiments that can be potentially standardized across laboratories worldwide.

Materials and Reagents

  1. 96-multiwell plates (Corning, Costar, catalog number: 2797)
  2. Kimwipes (Fisher, catalog number: 06-666A)
  3. 15 ml and 50 ml centrifuge tubes (MidsciTM, catalog numbers: 15 ml-CT2715, 50 ml-CT2750)
  4. Plastic domes (Hummert International, catalog number: 11-33480) and trays (Hummert International, catalog number: 11-33010)
  5. 9 cm round Petri dishes (Fisher, catalog number: FB0875712)
  6. 1.5 ml microfuge tubes (MidsciTM, catalog number: AVSS1700)
  7. Plastic pestles for 1.5 ml microfuge tubes (Fisher, catalog number: 12-141-364)
  8. 3 or 5 mm metal beads (QIAGEN, catalog number: 69997 or 69989)
  9. Trays for holding the Jiffy-7® peat pellets (Hummert International, catalog number: 11311000)
  10. 1 ml-syringes without needle (BD Biosciences, catalog number: 309659)
  11. Mesh bags (can be made by sewing a semi-oval out of a fine washable mesh material such as bridal veil; see Figure 3 for schematic; alternatively, perforated cellophane bread bags can be used)
  12. Tightly closing plastic container (with screwcap lid) 
  13. Damp paper towel 
  14. Fine bristled paint brush
  15. 96-well racked collection microtubes (optional, for 96-well tissuelyser format) (QIAGEN 19560)
  16. Arabidopsis thaliana (L. Heyhn.) ecotype Columbia (Col-0)
  17. Pseudomonas cannabina pv. alisalensis (formerly called P. syringae pv. maculicola ES4326 (Bull et al., 2010) carrying an empty vector (PmaDG3/Pma) (Guttman and Greenberg, 2001) bacterial culture (stored in 15% glycerol at -80 °C)
  18. Trichoplusia ni (eggs; Benzon Research or Natural Resources Canada)
  19. Pseudomonas simiae strain WCS417 (formerly called Pseudomonas fluorescens WCS417)
  20. Jiffy-7® peat pellets (Jiffy products, Canada, Hummert International, catalog number: 14-23700)
  21. Glycerol (Sigma-Aldrich, catalog number: G7757)
  22. K2HPO4 (Fisher, catalog number: P288)
  23. MgSO4 (Fisher, catalog number: BP213)
  24. Proteose peptone No. 3 (BD Biosciences-US, catalog number: 211693)
  25. Antibiotics
    1. Rifampicin (GoldBio, catalog number: R-120)
    2. Kanamycin (GoldBio, catalog number: K-120) 
  26. Agar (Fisher, catalog number: BP1423)
  27. Sterile distilled and tap water
  28. Bleach (Clorox Concentrated Germicidal Bleach)
  29. Ethanol (Decon Labs Inc., catalog number: 2701)
  30. Triton X-100 (Fisher, catalog number: BP151)
  31. Tryptone (BD Biosciences-US, catalog number: 211705)
  32. Sodium chloride (NaCl)
  33. Yeast extract (BD Biosciences-US, catalog number: 212750)
  34. 70% ethanol (see Recipes)
  35. 25% bleach supplemented with 0.1% Triton X-100 (see Recipes)
  36. 0.1% agar (see Recipes)
  37. King's medium B (KB) (see Recipes)
  38. Luria-Bertani medium (LB) (see Recipes)
  39. 10 mM MgSO4 (see Recipes)

Equipment

  1. Laboratory glassware
  2. 200 µl and 1 ml, and 20-200 µl multichannel micropipettes (Gilson)
  3. Forceps (Grainger, catalog number: 4CR15)
  4. 1 L beaker (Pyrex, 1000)
  5. Handheld electric drill (DeWALT, model: DWD110)
  6. Cork borer 4 mm diameter
  7. Autoclave (Primus Sterilizer Co. Inc. 1317. C.R.N: 09415.1256)
  8. Microcentrifuge (Eppendorf, model: 5415D)
  9. Freezer (-80 °C) (Panasonic VIP Plus, model: MDF-V76VC-PA)
  10. Plant Growth chamber at 20-23 °C with 12 h light/12 h dark (~75-100 μmol s-1m-2, cool white fluorescent; A1000 Conviron Growth Chamber with Arabidopsis Kit, or similar. Alternatively, a growth-room with ~135-145 μmol s-1m-2 -mix 50/50 of 400-watt sodium and metal halide bulbs or 75-100 μmol s-1m-2 cool white fluorescent bulbs can be used)
  11. Shaker incubator at 28 °C (Barnstead Max, model: Q 5000)
  12. Balance (Mettler Toledo, model: PB1501)
  13. Spectrophotometer (Bio-Mini SHIMADZU)
  14. Laminar flow hood (SterilGARD 3 Advance)
  15. Incubator at 28 °C (VWR, 3020)
  16. Analytical balance for weighing caterpillars 
  17. TissueLyser Beadmill (QIAGEN, catalog number: 85300) with 24 or 96 adapters (QIAGEN, catalog number: 69982 or 69984)

Procedure

  1. Hydration of Jiffy-7® peat pellets and Arabidopsis seed germination and growth (Figures 1A-1C)
    1. In 1.5 ml Eppendorf tubes, surface-sterilize 100-200 Arabidopsis seeds by washing with 70% ethanol for 2 min followed by 5 min with 25% bleach supplemented with 0.1% Triton X-100 and three washes in sterile water. Resuspend the seeds in 1-1.5 ml of a sterile solution of 0.1% agar (prepared in water and sterilized by autoclaving) using a pipette. Alternatively, chlorine gas sterilization can be used to eliminate endophytic bacteria, particularly to avoid microbial contamination for rhizosphere microbiome studies (Haney et al., 2015). 
    2. Prior to use, leave the seeds for 3-7 days at 4 °C to allow for their stratification. 
    3. Prepare 8-12 Jiffy-7® pellets for each genotype per bacterial infection experiment (or 25-30 pellets per treatment for T. ni assays, see Data analysis) by placing them in a germination tray.
    4. Hydrate Jiffy-7® peat pellets (Jiffy Products International, Canada) by immersing the base of the pellet with tap water. Allow the pellets to stand in water for at least 1 h to allow them to hydrate.
    5. Once the pellets are hydrated, use a pair of clean/sterile forceps to gently even/level the top surface of the pellet. 
    6. Sow 3-4 stratified seeds by pipetting them at the surface of each Jiffy-7® pellet and cover the trays with a plastic dome. Do not cover the seeds with soil.
    7. Transfer the trays to a growth chamber/room under 12 h day and 12 h night conditions at 23-20 °C day/night temperature regime, ~75-100 μmol s-1m-2 of cool white fluorescent light at rosette level, 50%-70% relative humidity. Alternatively, ~135-145 μmol s-1m-2 of mix 50/50 of sodium and metal halide light can be used.
      Note: Intensities of cool white fluorescence > 125 μmol s-1m-2 cause plants to bolt between 3 and 4 weeks and so should not be used.
    8. At 3-7 days post germination, carefully remove extra seedlings with clean/sterile forceps from the surface of the pellets leaving only a single seedling in each pellet (do not water seedlings during the first week after planting). 
    9. Two to three times per week, bottom water the Jiffy pellets by flooding the tray with tap water and pouring the excess water off after 30-60 min.

  2. P. simiae suspension preparation and seedling/pellets inoculation (Figures 1D-1F)
    1. When seedlings are eight days old, streak P. simiae WCS417 strain from a frozen glycerol stock onto a King’s B (KB) solid medium plate supplemented with Rifampicin (100 μg/ml). Allow it to grow for 24 h at 28 °C. Pick a single colony, streak it to a new plate and grow it for another 24 h.
    2. Inoculate 15 ml of media supplemented with Rifampicin (100 μg/ml) (in a 50 ml Falcon tube) with a loop of confluent bacteria from the plate. Grow in a 28 °C shaker incubator for 16-24 h. 
    3. Centrifuge the culture at 3,000 x g for 5 min, discard the supernatant and resuspend the pellet in 15 ml of sterile 10 mM MgSO4 solution. Repeat the centrifugation and finally resuspend the pellet in 15 ml 10 mM MgSO4 by pipetting up and down.
    4. Make a 1/10 or 1/20 dilution of the bacterial suspension (900 µl 10 mM MgSO4 + 100 µl bacterial suspension for 1:10 or 950 µl 10 mM MgSO4 + 50 µl bacterial suspension for 1:20 dilution) in a 1.5 ml tube and measure optical density at 600 nm (OD600) in a spectrophotometer to estimate the OD600 of the original P. simiae culture. (Divide the measured OD by the dilution factor). 
    5. In a clean (non-sterile) beaker, prepare at least 1 L of the P. simiae WCS417 inoculum by diluting the bacterial suspension culture in 10 mM MgSO4 to a final OD600 of 0.01. 
    6. Inoculate the ten-day-old seedlings with 6 ml of the bacterial solution per plant by gently pipetting on the top surface of the Jiffy-7® pellet, near the base of the seedling, taking care to avoid contact with the leaves or hypocotyl. Then, submerge the entire pellets for ~30 s in the same solution (using the 1 L beaker with the suspension) without allowing contact of the bacteria suspension with the seedling aerial tissues. One liter of bacterial solution can support the inoculation of at least 16 pellets. For the control/mock-treated plants follow the same steps but only using sterile MgSO4.
      Alternatively: If multiple strains are inoculated onto plants within the same flat, to avoid cross-contamination of bacterial strains between pellets, 2 ml of more concentrated inoculum (OD600 = 0.02-0.1) can be applied to the Jiffy pellets. For these assays, the pellets should be fairly dry (generally not watered after the initial soaking) and 2 days should pass after inoculation before flood watering the plants. Using this method, multiple bacterial strains and buffer-treated controls can be grown within the same flat with no detectable cross-contamination between pellets. Additionally, significantly smaller starting volumes of inoculum can be prepared.
    7. After these treatments, bottom water the plants as before for an additional 15-25 days (3.5-5 week-old plants) for Pma assays or 20 days (4.5 week-old plants) for T. ni assays.
      Note: Different genotypes can be planted side by side in the same tray. Rotate different trays 2-3 times per week to minimize growth variation.


    Figure 1. Schematic of P. simiae inoculation. A. Arrange Jiffy-7® peat pellets onto flats as shown. B. Flood the base of the trays with tap water for at least 60 min to allow the pellets to hydrate. C. Sow previously sterilized and stratified Arabidopsis seeds onto the surface of hydrated Jiffy® pellets. Water the pellets by flooding the base of the tray three times per week. D. Ten days after germination, inoculate 6 ml of P. simiae solution with OD600 = 0.01 onto the surface of the pellet taking care to avoid contact with the seedling aerial tissues. E. Immerse for ~30 s the Jiffy® pellet into a 1 L solution of P. simiae of OD600 = 0.01. One liter of bacterial solution can support inoculation of at least 16 pellets. F. Continue bottom-watering the pellets with tap water for a further 15 days (or 20 days for T. ni assay) until the plants are ready for plants infection with Pma or T. ni.

  3. Infections and growth of Pma or T. ni to analyze the effects of P. simiae WCS417 ISR
    To evaluate ISR against Pma (Figure 2)
    Growing Pma and infection
    1. Inoculate O/N cultures of Pseudomonas cannabina pv alisalensis carrying an empty vector (Pma) (Guttman and Greenberg, 2001) strain in KB-media supplemented with Kanamycin (50 μg/ml).
    2. The next morning, dilute the cultures back in KB medium supplemented with Kanamycin (1:5 ml) and grow for additional 3-4 h. 
    3. Spin the bacteria down at 3,000 x g for 3 min and resuspend in sterile 10 mM MgSO4. Repeat the wash an additional time. 
    4. Measure OD600 of the suspension by diluting 1:20 as described above in section B, point 4. Dilute the culture to an OD600 = 0.2 or 0.3 and dilute to a final OD600 = 0.0002-0.0003 by performing three 10-fold serial dilutions in sterile 10 mM MgSO4. The final volume should be 10 ml (~0.1 ml inoculum/leaf is required).
    5. Use a 1 ml syringe without a needle pressed up against the abaxial side of the 5th and 6th Arabidopsis leaves to infiltrate the bacteria into the apoplast of an entire leaf. Infect at least two leaves on eight different plants. Use a felt-tipped marker to mark the petiole of infected leaves for identification leading to the sampling and quantification next steps (see below).
      Note: Inoculate plants around 1 pm.
    6. With a Kimwipe, dry the excess bacterial suspension from each infiltrated leaf. Let the leaves dry for 1 h until the liquid in the apoplast is no longer visible. Then, cover the plants with a plastic dome to increase the humidity and reduce the variability in bacterial growth. Return plants to the growth chamber for 2-3 days.

    Quantifying bacterial growth in leaf tissues
    1. Two or three days after Pma inoculation, excise 8 leaf discs from different plants per treatment from infected leaves (ISR-induced vs. mock) using a cork borer (4 mm diameter). Take the discs from approximately the same leaf position for all the samples, 3-5 mm from the leaf tip. 
    2. Using forceps, place each leaf disc in a 1.5-ml microfuge tube containing 200 µl of 10 mM MgSO4.
    3. Grind the samples using a small hand-held electric drill with a plastic pestle. Thoroughly macerate the tissue until pieces of intact leaf tissue are no longer visible to the eye (avoiding heating up the samples by performing repeated short bursts of ~2 s instead of grinding continuously for a long-time interval). We suggest grinding each sample for a similar duration to minimize variability during the experiment. Samples can also be ground with the plastic pestle by hand (without the drill).
      Alternatively: Use a bead mill or TissueLyser to homogenize the samples. To use a TissueLyser, place the leaf disk into a 2 ml Eppendorf tube with a 5 mm metal bead and 100 µl water. Alternatively, a 96-well plate format can be used with 3 mm beads. Homogenize tissue for 2 min at 25 Hz.
    4. Next, vortex the homogenate and remove 20 µl from each sample and dilute in 180 µl 10 mM MgSO4 in 96-well multi-well plate. Use a multichannel pipette to repeat this process 4 times to have a serial 1:10 dilution series (10-1 to 10-5) for each sample. 
    5. Plate 20 µl aliquots of the 1:10 serial dilutions (10-2 to 10-5) on KB medium plates supplemented with Kanamycin (50 μg/ml of media) and allowed to dry onto the surface. Divide each KB plate with a marker such that at least eight samples/dilutions per plate can be plated.
      Alternatively: By pouring media into rectangular plates, 10 µl can be spotted with a multichannel pipette, and all samples can be plated on a single plate (see Figure 2). 
    6. Incubate the plates at 28 °C for 2-3 days until colony-forming units (CFU) can be counted. Calculate the number of CFU per leaf disc by multiplying by the corresponding dilution factor (see Data analysis).


      Figure 2. Images showing the key steps to evaluate ISR against Pma in Arabidopsis grown in Jiffy-7® peat pellets. A. Infiltrate the bacteria suspension into the abaxial surface of Arabidopsis leaves with a 1 ml-syringe without a needle (Pma, OD600 = 0.0003) (Note: Inoculate plants at ~1 p.m.). B. Two-three days after Pma infection, excise leaf discs from the infiltrated leaves using a cork borer (4 mm diameter). C. Place each leaf disc in a 1.5-ml microfuge tube with 200 µl of 10 mM MgSO4 solution. D. Grind the samples using a plastic pestle mounted in a hand-held electric drill (shown) or a bead beater/TissueLyser (not shown). E. Grind the leaf discs until the tissue is thoroughly homogenized and no visible pieces of tissue remain (Note: Avoid heating up the samples by performing ~2 s bursts). F. Remove 20 µl from each sample and dilute in 180 µl 10 mM MgSO4 in a multi-well plate and repeat this process to have a 1:10 dilution series (10-1 to 10-5) for each sample. Plate 20 µl aliquots of each dilution on KB medium plates or 10 µl aliquots onto rectangular plates.

    To evaluate ISR against Trichoplusia ni (Figure 3)
    1. Incubate T. ni eggs (Benzon Research or Natural Resources Canada) at 30 °C for 36 h with 12-h light days. The photoperiod of the chamber should be in synch with that of the plants so the caterpillars are entrained with the same photoperiod.
    2. Using a minimum of 25 plants per treatment at 4-5 weeks of age, randomly choose one newly hatched caterpillar larva from the batch. Using a small paint brush, place one larva at the center of the rosette of a Jiffy-7® pellet grown Arabidopsis rosette pretreated with Pseudomonas or buffer control.
    3. Cover each pellet/plant with a mesh bag (Figure 3), return to a growth chamber, and allow the caterpillars to feed for 7 days. Plants should be at a density of no more than 30 per flat.
    4. On the 7th day, remove the mesh bag and find the caterpillar on the plant. Weigh the larva with a precision balance to the nearest tenth of a milligram. The weight of newly hatched larvae is negligible; thus, the final caterpillar weight correlates with how much the caterpillar ate and host plant susceptibility (Cui et al., 2002).


    Figure 3. T. ni herbivory assay. A. Mesh bags can be made by cutting mesh (see materials) in a half-oval of ~200 x 200 mm and then using a sewing machine to sew two half-ovals together (dashed line). B. Place cheese cloth with T. ni eggs in a tightly sealing container with a damp paper towel at the bottom. Place the container in an incubator at 30 °C with the same light regime as the plants until the larvae hatch (~24-36 h). C. Using a fine paint brush, transfer a small number of newly hatch T. ni larvae to a Petri plate or other small container. Reseal the original container. Using the paint brush, place 1 larva on the center of the rosette of a 4-5 week-old Arabidopsis plant. D. Cover each plant with a mesh bag and secure at the base with a rubber band. Return the plants to the flats and return flats to the incubator. Allow caterpillars to feed for 1 week. E. One week later, find and weigh each individually (to the nearest 0.1 mg).

Data analysis

ISR-Pma data analysis
When the colonies are still small, count the CFUs for one of the dilutions (where 10-50 CFU can be counted and colonies are clearly distinct) for each disc from one leaf per plant taken and estimate the number of bacteria by multiplying with the corresponding dilution factor. Perform CFU counting for at least eight discs (12 discs provide greater statistical power and allows for increased confidence in smaller differences in bacterial growth). To increase confidence in possible differences in growth, replicate the entire experiment at least 3 independent times on different days and from different batches of inoculum. Use all the data collected from all independent experiments performed to calculate the average CFU per leaf disc plus/minus standard error. To determine if differences are statistically significant, perform analysis of variance (ANOVA) and a post-hoc test such Tukey’s HSD test or Newman-Keuls (SNK) by using appropriate statistical software (Figure 4D and methods in Cecchini et al., 2019). If only two conditions are being compared, a Student’s t-test can be used. Plot the data on a log10 scale.

ISR-T. ni data analysis
Weigh at least 25 larvae/plants per treatment, per experiment and repeat the experiment a minimum of 3 independent times. Use all the data collected from all independent experiments performed to calculate the average weight per larvae plus/minus standard error. To determine if differences are statistically significant, perform analysis of variance (ANOVA) and a post hoc test such Tukey’s HSD test or Newman-Keuls (SNK) by using appropriate statistical software. If only two conditions are being compared, a Student’s t-test can be used. If the experiments are done in different chambers or with insects from different sources, a significant replicate effect has been observed (Haney et al., 2018). As a result, data can be normalized to the buffer-treated Col-0 control data from a single experiment prior to averaging the data from at least 3 independent experiments.

Notes

Using Jiffy-7® has allowed us to study interactions between diverse below and above ground commensals and pathogens. This setup has been used to study diverse rhizosphere bacteria that induce systemic resistance or susceptibility (Haney et al., 2015 and 2018; Melnyk et al., 2019). Additionally, it can be used to study below ground effects on diverse above ground pathogens including pathogenic Pseudomonas and Xanthomonas spp. (Haney et al., 2018) and fungal pathogens such as Botrytis, and downy mildew.

Recipes

  1. 70% ethanol
    73.7 ml of 95% ethanol
    Add distilled water up to 100 ml
  2. 25% bleach supplemented with 0.1% Triton X-100
    25 ml bleach and 100 µl of Triton X-100
    Add distilled water to 100 ml
  3. 0.1% agar
    Dissolve 0.1 g agar in 100 ml sterile distilled water by autoclaving, swirl the solution while it cools
  4. King's medium B (KB)
    20 g of proteose peptone No. 3
    10 g glycerol
    1.5 g MgSO4
    1.2 g K2HPO4
    For solid medium add 13 g agar
    Add distilled water up to 1 L
    Sterilize by autoclaving
  5. Luria-Bertani medium (LB)
    10 g tryptone
    10 g sodium chloride (NaCl)
    5 g yeast extract
    Add distilled water up to 1 L
    Sterilize by autoclaving
  6. 10 mM MgSO4
    0.12 g of MgSO4
    Add distilled water to 100 ml
    Sterilize by autoclaving

Acknowledgments

We thank Elizabeth Baldo and Joanna Jelenska for helping identify catalog numbers for materials and equipment.
  This work was supported by NSF grant IOS1456904 to JTG, by an NSERC Discovery Grant (NSERCRGPIN-2016-04121) awarded to CHH, a China Postdoctoral Science Foundation Fellowship awarded to YS and an Agencia Nacional de Promoción Científica y Tecnológica grant (PICT-2017-0589) awarded to NMC. NMC is a Career Investigator of CONICET (Argentina).

Competing interests

The authors declare that they have no conflict of interests.

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  17. Pozo, M. J., Van Der Ent, S., Van Loon, L. C. and Pieterse, C. M. (2008). Transcription factor MYC2 is involved in priming for enhanced defense during rhizobacteria-induced systemic resistance in Arabidopsis thaliana. New Phytol 180(2): 511-523.
  18. Verhagen, B. W., Glazebrook, J., Zhu, T., Chang, H. S., van Loon, L. C. and Pieterse, C. M. (2004). The transcriptome of rhizobacteria-induced systemic resistance in Arabidopsis. Mol Plant Microbe Interact 17(8): 895-908.

简介

摘要:植物免疫系统对于植物感知和防御细菌,真菌和昆虫害虫和病原体是必不可少的。诱导的系统抗性(ISR)是在有益微生物的根定殖时发生的全身免疫应答。一项经过充分研究的ISR模型是 Pseudomonas spp的特定有益菌株的关联。与参考植物拟南芥。在这里,我们描述了一种强大的,增加的通量,生物测定,以研究针对细菌病原体 Pseudomonas cannabina pv的ISR。 alisalensis (以前称为 Pseudomonas syringae pv.maculicola )菌株ES4326和草食动物 Trichoplusia ni 通过接种在Jiffy-7 ®泥炭颗粒中生长的拟南芥植物上的假单胞菌菌株WCS417(以前称为荧光假单胞菌 WCS417)。虽然最常用于 Pseudomonas -triggered ISR on 拟南芥,但该测定对多种根际细菌菌株,植物物种,病原体和食草动物有效。

背景:植物感染或通过病原微生物或共生微生物定植后,可发生涉及免疫“记忆”或“引发”的系统防御反应(Parker,2009; Fu and Dong,2013; Martinez-Medina et al。 ,2016)。根据微生物和相关的植物器官,诱导了不同的系统抗性程序(Pieterse et al。,2014)。当根遇到特定的共生微生物时,观察到远端根和芽组织中诱导的系统抗性(ISR)(Pieterse et al。,1996; van Loon et al。,1998 )。尽管在ISR反应期间部署的遗传和激素信号传导机制已得到部分了解(Verhagen et al。,2004; Pieterse et al。,2014; Pangesti et al 。,2016),许多问题仍未得到解决,例如根 - 微生物组通信中涉及的机制或系统信号的身份(Pieterse 等。,2014; Haney et al。,2018; Cecchini et al。,2019)。

Pseudomonas simiae WCS417接种拟南芥根已被用作研究针对细菌,真菌和食草动物入侵者的ISR的模型系统(Pieterse et al。,1996; van Loon et al。,1998; Verhagen et al。,2004; Haney et al。,2015)。现有的 P生物测定法。 simiae - 拟南芥 ISR利用无菌沙发芽拟南芥,然后将幼苗移植到含有根际细菌的沙/土壤混合物中(Pieterse et al 。,1996; Pozo et al。,2008; van Oosten et al。,2008)。基于这种方法,我们最近开发了针对细菌病原体 Pseudomonas cannabina pv的ISR生物测定法。 alisalensis (以前称为 Pseudomonas syringae pv。 maculicola )菌株ES4326( Pma )和草食动物 Trichoplusia ni T.ni )使用拟南芥植物发芽并在Jiffy-7®泥炭颗粒中生长。这是一个有效的系统,可以有力地触发和量化ISR,主要是因为泥炭颗粒具有恒定的体积和低荧光假单胞菌含量(Haney et al。,2015和2018; Cecchini et al。,2019)。此外,避免幼苗移植减少了实验劳动力和对幼苗的压力,从而提高了测定效率。在这里,我们描述了ISR实验的逐步生物测定方法,该方法可以在全球实验室中进行潜在的标准化。

关键字:诱导系统抗性, 猴假单胞菌, 拟南芥, 丁香假单胞菌, 大麻假单胞菌, 粉纹夜蛾

材料和试剂

  1. 96孔板(Corning,Costar,目录号:2797)
  2. Kimwipes(Fisher,目录号:06-666A)
  3. 15 ml和50 ml离心管(Midsci TM ,目录号:15 ml-CT2715,50 ml-CT2750)
  4. 塑料圆顶(Hummert International,目录号:11-33480)和托盘(Hummert International,目录号:11-33010)
  5. 9厘米圆形培养皿(Fisher,目录号:FB0875712)
  6. 1.5 ml微量离心管(Midsci TM ,目录号:AVSS1700)
  7. 用于1.5 ml微量离心管的塑料杵(Fisher,目录号:12-141-364)
  8. 3或5毫米金属珠(QIAGEN,目录号:69997或69989)
  9. 用于容纳Jiffy-7 ®泥炭颗粒的托盘(Hummert International,目录号:11311000)
  10. 1毫升无针注射器(BD Biosciences,目录号:309659)
  11. 网袋(可以通过用可洗的细网状材料如新娘面纱缝制半椭圆形来制作;参见图3的示意图;或者,可以使用穿孔的玻璃纸面包袋)
  12. 紧紧关闭塑料容器(带螺旋盖) 
  13. 湿纸巾 
  14. 细毛漆刷
  15. 96孔齿条收集微管(可选,用于96孔组织器格式)(QIAGEN 19560)
  16. 拟南芥(L。Heyhn。)生态型哥伦比亚(Col-0)
  17. Pseudomonas cannabina pv。 alisalensis (以前称为 P.syringae pv。 maculicola ES4326(Bull et al。,2010)携带空载体( Pma DG3 / Pma )(Guttman和Greenberg,2001)细菌培养物(在-80°C下储存在15%甘油中)
  18. Trichoplusia ni (鸡蛋; Benzon研究或加拿大自然资源部)
  19. Pseudomonas simiae 菌株WCS417(以前称为 Pseudomonas fluorescens WCS417)
  20. Jiffy-7 ®泥炭颗粒(Jiffy products,Canada,Hummert International,目录号:14-23700)
  21. 甘油(Sigma-Aldrich,目录号:G7757)
  22. K 2 HPO 4 (Fisher,目录号:P288)
  23. MgSO 4 (Fisher,目录号:BP213)
  24. 蛋白胨第3号(BD Biosciences-US,目录号:211693)
  25. 抗生素
    1. 利福平(GoldBio,目录号:R-120)
    2. 卡那霉素(GoldBio,目录号:K-120) 
  26. 琼脂(Fisher,目录号:BP1423)
  27. 无菌蒸馏水和自来水
  28. 漂白剂(Clorox浓缩杀菌剂)
  29. 乙醇(Decon Labs Inc.,目录号:2701)
  30. Triton X-100(Fisher,目录号:BP151)
  31. 胰蛋白胨(BD Biosciences-US,目录号:211705)
  32. 氯化钠(NaCl)
  33. 酵母提取物(BD Biosciences-US,目录号:212750)
  34. 70%乙醇(见食谱)
  35. 25%漂白剂补充0.1%Triton X-100(见食谱)
  36. 0.1%琼脂(见食谱)
  37. 国王的中等B(KB)(见食谱)
  38. Luria-Bertani培养基(LB)(见食谱)
  39. 10 mM MgSO 4 (见食谱)

设备

  1. 实验室玻璃器皿
  2. 200μl和1 ml,以及20-200μl多通道微量移液管(Gilson)
  3. 镊子(Grainger,目录号:4CR15)
  4. 1升烧杯(Pyrex,1000)
  5. 手持式电钻(DeWALT,型号:DWD110)
  6. 软木钻孔机直径4毫米
  7. 高压灭菌器(Primus Sterilizer Co. Inc. 1317. C.R.N:09415.1256)
  8. 微量离心机(Eppendorf,型号:5415D)
  9. 冷冻箱(-80°C)(Panasonic VIP Plus,型号:MDF-V76VC-PA)
  10. 植物生长室在20-23°C,12小时光照/ 12小时黑暗(~75-100μmols -1 m -2 ,冷白色荧光; A1000 Conviron具有拟南芥试剂盒或类似物的生长室。或者,生长室~135-145μmols -1 m -2 -mix可以使用50/50的400瓦钠和金属卤化物灯泡或75-100μmols -1 m -2 冷白色荧光灯泡)
  11. 振荡器培养箱温度为28°C(Barnstead Max,型号:Q 5000)
  12. Balance(Mettler Toledo,型号:PB1501)
  13. 分光光度计(Bio-Mini SHIMADZU)
  14. 层流罩(SterilGARD 3 Advance)
  15. 孵化器温度为28°C(VWR,3020)
  16. 称重毛毛虫的分析天平 
  17. TissueLyser Beadmill(QIAGEN,目录号:85300),带24或96个适配器(QIAGEN,目录号:69982或69984)

程序

  1. Jiffy-7 ®泥炭颗粒的水合作用和拟南芥种子的萌发和生长(图1A-1C)
    1. 在1.5ml Eppendorf管中,通过用70%乙醇洗涤2分钟对100-200个拟南芥种子进行表面灭菌,然后用25%漂白剂补充0.1%Triton X-100并在三次洗涤中洗涤5分钟。无菌水。使用移液管将种子重悬于1-1.5ml 0.1%琼脂的无菌溶液(在水中制备并通过高压灭菌灭菌)。或者,氯气灭菌可用于消除内生细菌,特别是避免微生物污染根际微生物组研究(Haney et al。,2015)。 
    2. 使用前,将种子在4°C下放置3-7天,以便进行分层。 
    3. 每个细菌感染实验为每种基因型准备8-12个Jiffy-7 ®颗粒(或者每个处理25-30个颗粒用于 T.ni 分析,参见数据分析)它们在发芽盘中。
    4. 通过用自来水浸泡颗粒的基质来水合Jiffy-7 ®泥炭颗粒(Jiffy Products International,Canada)。使颗粒在水中静置至少1小时以使其水合。
    5. 一旦颗粒水合,使用一对干净/无菌的镊子轻轻均匀/平整颗粒的顶部表面。 
    6. 通过在每个Jiffy-7 ®颗粒的表面移液,播种3-4个分层种子,并用塑料圆顶覆盖托盘。不要用土壤覆盖种子。
    7. 将托盘在12小时和12小时夜间条件下转移到生长室/室中,在23-20℃昼/夜温度下,~75-100μmols -1 m - 莲座级冷白色荧光灯,相对湿度50%-70%。或者,可以使用约135-145μmols -1 m -2 的混合物50/50的钠和金属卤化物光。
      注意:冷白色荧光的强度> 125μmols -1 m -2 导致植物在3到4周之间停植,因此不应使用。
    8. 发芽后3-7天,用颗粒表面的清洁/无菌镊子小心地去除多余的幼苗,每个颗粒中只留下一个幼苗(种植后第一周不要浇水幼苗)。 
    9. 每周两到三次,通过用自来水淹没托盘并在30-60分钟后倒掉多余的水来底部浇注Jiffy颗粒。

  2. P上。 simiae 悬浮液制备和幼苗/颗粒接种(图1D-1F)
    1. 幼苗8天时,条纹 P. simiae WCS417菌株从冷冻甘油原液到补充有利福平(100μg/ ml)的King's B(KB)固体培养基平板上。让它在28°C下生长24小时。选择一个单一的菌落,将其连接到一个新的平板并再生长24小时。
    2. 接种15ml补充有利福平(100μg/ ml)(在50ml Falcon管中)的培养基,其中含有来自培养板的汇合细菌环。在28°C摇床培养箱中培养16-24小时。 
    3. 将培养物在3,000μL离心下离心5分钟,弃去上清液并将沉淀重悬于15ml无菌10mM MgSO 4 溶液中。重复离心,最后通过上下吸移将沉淀重悬于15ml 10mM MgSO 4 中。
    4. 制备1/10或1/20稀释的细菌悬液(900μl10mM MgSO 4 +100μl细菌悬液1:10或950μl10mM MgSO 4 在1.5 ml管中加入50μl细菌悬液(1:20稀释),并在分光光度计中测量600 nm处的光密度(OD 600 )以估算OD 600 原来的 P. simiae 文化。 (将测得的OD除以稀释系数)。 
    5. 在干净(非无菌)烧杯中,准备至少1L的 P.通过将细菌悬浮培养物在10mM MgSO 4 中稀释至最终OD 600 0.01来接种WCS417接种物。 
    6. 通过在幼苗基部附近的Jiffy-7 颗粒的顶部表面轻轻移液,每株植物接种10天的幼苗和6ml细菌溶液接种,注意避免与叶子或下胚轴接触。然后,将整个颗粒浸没在同一溶液中约30秒(使用带有悬浮液的1L烧杯),不允许细菌悬浮液与幼苗空气组织接触。一升细菌溶液可以支持接种至少16个颗粒。对于对照/模拟处理的植物,遵循相同的步骤,但仅使用无菌的MgSO 4 。
      或者:如果将多个菌株接种到同一个单位内的植物上,以避免细菌菌株在颗粒之间交叉污染,则加入2 ml浓度更高的接种物(OD 600 = 0.02-0.1)可应用于Jiffy颗粒。对于这些测定,颗粒应该相当干燥(通常在初始浸泡后不浇水)并且在接种之前应该通过2天,然后给植物浇水。使用该方法,可以在同一平面内生长多个细菌菌株和缓冲液处理的对照,在颗粒之间没有可检测的交叉污染。另外,可以制备明显更小的接种物起始体积。
    7. 在这些处理之后,如前所述给植物底部浇水另外15-25天(3.5-5周龄植物)用于 Pma 测定或20天(4.5周龄植物)用于 T. ni 检测。
      注意:可以在同一个托盘中并排种植不同的基因型。每周旋转不同的托盘2-3次,以尽量减少生长变化。


    图1. P的示意图。 simiae 接种。 A.如图所示,将Jiffy-7 ®泥炭颗粒放在平面上。 B.用自来水将托盘底部浸泡至少60分钟,以使颗粒水合。 C.播种先前已经灭菌并将拟南芥种子分层到水合Jiffy ®颗粒的表面上。通过每周三次淹没托盘底部来浇水颗粒。 D.发芽后10天,接种6ml P.将OD 600 = 0.01的simiae 溶液置于颗粒表面,注意避免与幼苗空气组织接触。 E.将约30秒的Jiffy ®颗粒浸入1L的 P溶液中。 OD 600 = 0.01的simiae 。 1升细菌溶液可以支持接种至少16个颗粒。 F.继续用自来水对颗粒进行底部浇水15天(或 T.ni 测定20天),直到植物准备用 Pma 进行植物感染。或者 T.你。

  3. Pma 或 T的感染和生长。 ni 分析 P的影响。 simiae WCS417 ISR
    评估ISR Pma (图2)
    增长 Pma 和感染
    1. 在KB培养基中接种携带空载体( Pma )(Guttman和Greenberg,2001)菌株的 Pseudomonas cannabina pv alisalensis 的O / N培养物补充卡那霉素(50μg/ ml)。
    2. 第二天早上,将培养物稀释在补充有卡那霉素(1:5 ml)的KB培养基中,再生长3-4小时。 
    3. 将细菌在3,000 x g 下旋转3分钟并重悬于无菌的10mM MgSO 4 中。再重复一次洗涤。 
    4. 如上文B部分第4点所述,通过稀释1:20测量悬浮液的OD 600 。将培养物稀释至OD 600 = 0.2或0.3并稀释至通过在无菌10mM MgSO 4 中进行三次10倍连续稀释,最终OD 600 = 0.0002-0.0003。最终体积应为10毫升(需要约0.1毫升接种物/叶)。
    5. 使用1毫升注射器,不用针头压在5 和6 <


      拟南芥叶的背面,将细菌渗入细菌整片叶子的质外体。在八种不同的植物上感染至少两片叶子。使用毡尖标记来标记受感染叶子的叶柄以进行鉴定,从而导致采样和量化后续步骤(见下文)。
      注意:下午1点左右接种植物。
    6. 使用Kimwipe,从每个渗透的叶子中干燥多余的细菌悬浮液。让叶子干燥1小时,直到质外体中的液体不再可见。然后,用塑料圆顶覆盖植物,以增加湿度并减少细菌生长的变化。将植物返回生长室2-3天。

    量化叶组织中的细菌生长
    1. Pma 接种后2或3天,使用软木钻孔器(直径4mm)从感染的叶子(ISR诱导对模拟)中每次处理从不同植物中切下8个叶盘。从所有样品中取出大致相同叶片位置的圆盘,距离叶尖3-5毫米。&nbsp;
    2. 使用镊子,将每个叶盘置于含有200μl10mMMgSO 4 的1.5ml微量离心管中。
    3. 使用带有塑料杵的小型手持式电钻研磨样品。彻底浸渍组织,直到眼睛看不到完整的叶片组织(避免通过重复短时间的~2秒的爆发而不是长时间连续研磨来加热样品)。我们建议将每个样品研磨相似的持续时间,以尽量减少实验过程中的变异性。样品也可以用塑料研杵手工研磨(不用钻头)。
      或者:使用珠磨机或TissueLyser均化样品。要使用TissueLyser,将叶盘放入带有5 mm金属珠和100μl水的2 ml Eppendorf管中。或者,96孔板形式可以与3mm珠子一起使用。将组织在25Hz下均化2分钟。
    4. 接下来,涡旋匀浆并从每个样品中移除20μl,并在96孔多孔板中在180μl10mMMgSO 4 中稀释。使用多通道移液器重复此过程4次,以获得每个样品的1:10稀释系列(10 -1 至10 -5 )。&nbsp;
    5. 在补充有卡那霉素(50μg/ ml培养基)的KB培养基平板上平板加入20μl等份的1:10系列稀释液(10 -2 至10 -5 ),允许干燥到表面上。将每个KB板用标记分开,使得每个板至少可以镀8个样品/稀释液。
      或者:通过将培养基倒入矩形板中,可以用多通道移液器点样10μl,所有样品都可以镀在一个平板上(见图2)。
    6. 将板在28℃孵育2-3天,直到可以计数菌落形成单位(CFU)。通过乘以相应的稀释因子计算每片叶片的CFU数量(参见数据分析)。


      图2.显示在Jiffy-7 ® 泥炭颗粒。 A.用1 ml注射器将细菌悬浮液浸入拟南芥叶的背轴表面针( Pma ,OD 600 = 0.0003)(注意:在~1 pm 接种植物)。 B. Pma 感染后2-3天,使用软木钻孔器(直径4mm)从渗入的叶子中切下叶盘。 C.将每个叶盘置于具有200μl10mMMgSO 4 溶液的1.5ml微量离心管中。 D.使用安装在手持式电钻(图示)或珠磨机/ TissueLyser(未示出)中的塑料研磨物研磨样品。 E.研磨叶盘,直到组织完全均质化,并且没有可见的组织残留物(注意:避免通过~2秒的爆发来加热样品)。 F.从每个样品中取出20μl并在多孔板中稀释于180μl10mMMgSO 4 中并重复该过程以具有1:10稀释系列(10 -1 < / sup>至10 -5 )对于每个样品。将每种稀释液的20μl等分试样在KB培养基平板上或10μl等分试样平板放置在矩形平板上。

    评估ISR Trichoplusia ni (图3)
    1. 孵育 T. ni 鸡蛋(Benzon Research或Natural Resources Canada)在30°C下36小时,12小时光照天。室的光周期应该与植物的光周期同步,因此毛虫被相同的光周期所夹带。
    2. 在4-5周龄时每次处理至少使用25株植物,随机选择一批新孵出的毛虫幼虫。使用小刷子,将一只幼虫放在用 Pseudomonas 预处理的Jiffy-7 ®颗粒生长的拟南芥莲座丛中心或缓冲控制。
    3. 用网袋覆盖每个颗粒/植物(图3),返回生长室,让毛虫喂食7天。植物的密度应不超过每平30个。
    4. 在7 th 日,取下网袋,找到植物上的毛毛虫。用精确的天平称重幼虫,精确到十分之一毫克。新孵化幼虫的重量可以忽略不计;因此,最终的毛虫重量与毛虫吃多少和寄主植物敏感性相关(Cui et al。,2002)。


    图3. T. ni 草食性试验。 A.网袋可以通过切割网眼(见材料)制成半椭圆形~200 x 200 mm,然后用缝纫机将两个半椭圆缝合在一起(虚线)。 B.用 T放置奶酪布。 ni 将鸡蛋放在密封的容器中,底部有湿纸巾。将容器置于30°C的培养箱中,其光照条件与植物相同,直至幼虫孵化(~24-36小时)。 C.使用精细的油漆刷,转移少量新孵化的 T. ni 幼虫到培养皿或其他小容器中。重新密封原始容器。使用油漆刷,将1只幼虫放在4-5周龄的拟南芥植物的莲座中心。 D.用网袋覆盖每个植物,并用橡皮筋固定在基部。将植物放回平底板并将平板放回培养箱。让毛毛虫喂食1周。 E.一周后,单独找到并称重(精确到0.1毫克)。

数据分析


当菌落仍然很小时,从每株植物的一片叶子中计算其中一个稀释液(其中10-50 CFU可以计数,菌落明显不同)的CFU,并通过乘以相应的数量估算细菌数量。稀释因子。对至少8个圆盘进行CFU计数(12个圆盘提供更大的统计功效,并且可以增加细菌生长中较小差异的可信度)。为了增加对可能的生长差异的信心,在不同日期和不同批次的接种物中至少复制整个实验至少3次。使用从所有独立实验中收集的所有数据来计算每叶片平均CFU加/减标准误差。为了确定差异是否具有统计学意义,通过使用适当的统计软件(图4D和Cecchini等人的方法),进行方差分析(ANOVA)和事后检验,如Tukey's HSD检验或Newman-Keuls(SNK)。 ,2019)。如果仅比较两个条件,则可以使用学生的 t - 测试。在log 10 比例上绘制数据。

每次实验每次处理称重至少25个幼虫/植物,并至少重复实验3次。使用从所有独立实验中收集的所有数据来计算每个幼虫的平均重量加/减标准误差。为了确定差异是否具有统计学意义,通过使用适当的统计软件进行方差分析(ANOVA)和事后检验,如Tukey's HSD检验或Newman-Keuls(SNK)。如果仅比较两个条件,则可以使用学生的 t - 测试。如果实验是在不同的室中进行的,或者是来自不同来源的昆虫,则观察到显着的重复效应(Haney et al。,2018)。结果,在平均来自至少3次独立实验的数据之前,可以将数据标准化为来自单次实验的缓冲液处理的Col-0对照数据。

笔记

使用Jiffy-7 ®使我们能够研究不同地下和地上共生物和病原体之间的相互作用。该设置已被用于研究诱导系统抗性或易感性的多种根际细菌(Haney 等人,2015和2018; Melnyk 等人,,2019)。此外,它可用于研究地下多种病原体的地下影响,包括病原性 Pseudomonas 和 Xanthomonas spp。 (Haney et al。,2018)和真菌病原体如 Botrytis 和霜霉病。

食谱

  1. 70%乙醇
    73.7毫升95%乙醇
    加入蒸馏水至100毫升
  2. 25%漂白剂补充0.1%Triton X-100
    25毫升漂白剂和100微升Triton X-100
    加入蒸馏水至100毫升
  3. 0.1%琼脂
    通过高压灭菌将0.1g琼脂溶解在100ml无菌蒸馏水中,在溶液冷却时旋转溶液
  4. 国王的中等B(KB)
    20克蛋白胨第3号
    10克甘油
    1.5克MgSO 4
    1.2克K 2 HPO 4
    对于固体培养基,加入13克琼脂
    加入蒸馏水至1升
    通过高压灭菌消毒
  5. Luria-Bertani中等(LB)
    10克胰蛋白胨
    10克氯化钠(NaCl)
    5克酵母提取物
    加入蒸馏水至1升
    通过高压灭菌消毒
  6. 10mM MgSO 4
    0.12克MgSO 4
    加蒸馏水至100毫升
    通过高压灭菌消毒

致谢

我们感谢Elizabeth Baldo和Joanna Jelenska帮助确定材料和设备的目录号。
&NBSP;这项工作得到了NSF资助IOS1456904到JTG的支持,NSERC发现资助(NSERCRGPIN-2016-04121)授予CHH,中国博士后科学基金奖学金授予YS和Agencia NacionaldePromociónCientíficayTecnológica资助(PICT-2017) -0589)授予NMC。 NMC是CONICET(阿根廷)的职业调查员。

利益争夺

作者声明他们没有利益冲突。

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引用:Cecchini, N. M., Song, Y., Roychoudhry, S., Greenberg, J. T. and Haney, C. H. (2019). An Improved Bioassay to Study Arabidopsis Induced Systemic Resistance (ISR) Against Bacterial Pathogens and Insect Pests. Bio-protocol 9(10): e3236. DOI: 10.21769/BioProtoc.3236.
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