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Establishment of a Fusarium graminearum Infection Model in Arabidopsis thaliana Leaves and Floral Tissues
建立拟南芥叶片和花组织的禾谷镰刀菌感染模型   

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Abstract

Fusarium graminearum (Fg) is the causal agent of Fusarium head blight disease of wheat (Triticum aestivum), oats (Avena sativa) and barley (Hordeum vulgare), which targets the floral tissues and thereby adversely impacts grain yield and quality. Mycotoxins produced by F. graminearum further limit the consumability of infected grain. In the laboratory, F. graminearum also has the ability to colonize both leaves and inflorescence tissues of Arabidopsis thaliana. The interaction between A. thaliana and F. graminearum makes available a large array of genetic and molecular tools to study the interaction between plants and F. graminearum to elucidate plant genes and pathways that contribute to resistance, as well as study how the fungus targets plant genes and mechanisms to promote disease. The methods described below allow for efficient infection of Arabidopsis leaves and inflorescence, and evaluation of disease progress and fungal growth. Disease spread in Arabidopsis can be readily monitored by the visual observations of chlorosis of leaf tissue and disease phenotype of inflorescence tissue including fungal mass on surface of the inflorescence tissue. Fungal growth can be further monitored by measuring the relative amount of Fg DNA in the host tissue by polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR).

Materials and Reagents

  1. PCR tubes (Fisher Scientific, catalog number: 14222 262 )
  2. Petri dishes (100 x 15 mm) (Fisher Scientific, catalog number: FB0875713 )
  3. 50 ml plastic screw-capped tubes (Midsci, catalog number: C50B )
  4. Pipette tips (sterile) (Midsci, catalog number: AVR-1, AVR-4 and AVR-11 )
  5. 1.7 ml microfuge tubes (sterile) (Catalog number: AVSS1700 )
  6. Cheesecloth from a local craft store or Miracloth (EMD Millipore, catalog number: 475855-1R )
  7. Culture tube
  8. 1 ml needle-less syringe (Tuberculin syringe) (Becton Dickinson, catalog number: 309659 )
  9. Funnel
  10. 1 L glass conical flask (Pyrex brand)
  11. Tweezers
  12. Hemocytometer
  13. Camel hair brush
  14. Sharpie or comparable water-proof marker
  15. Disposable gloves
  16. Kimwipes, tissue paper or paper towels
  17. Face shield (Fisher Scientific, catalog number: 18-999-4542 )
  18. Kord brand 3.5 inch square pots with bottom holes (Hummert International, catalog number: 12-1350-1 )
  19. T.O. Plastics Standard Flats 1020 tray with bottom holes (Hummert International, catalog number: 11-3000-1 )
  20. T.O. Plastics Standard Flats 1020 tray without holes (Hummert International, catalog number: 11-3050-1 )
  21. DOM1020 plastic dome to fit 1020 flats (Hummert International, catalog number: 11-3360-1 )
  22. Transparent plastic bags (Glad 13 gallon Recycling Drawstring Clear Trash bag)
  23. Fusarium graminearum isolate Z-3639 (Bowden and Leslie, 1999)
  24. Arabidopsis thaliana seeds (Accession Columbia, Nössen, and Wassilewskija)
  25. Silwet L-77 (Lehle seeds, catalog number: VIS-30 )
  26. Potato Dextrose Broth (Becton Dickinson, catalog number: 254920 )
  27. Yeast extract (Becton Dickinson, catalog number: 212750 )
  28. BD Difco Agar (Becton Dickinson, catalog number: 214530 )
  29. Ammonium Nitrate (Fisher Scientific, catalog number: A676 )
  30. Potassium chloride (Fisher Scientific, catalog number: P217 )
  31. Magnesium sulfate heptahydrate (Fisher Scientific, catalog number: M63 )
  32. Sodium chloride (Fisher Scientific, catalog number: BP358-1 )
  33. Tris-Base (Fisher Scientific, catalog number: BP152 )
  34. Ethylenediaminetetraacetic acid, disodium salt, Dihydrate (Fisher Scientific, catalog number: S311 )
  35. Sodium dodecyl sulfate (Fisher Scientific, catalog number: BP166 )
  36. Carboxymethyl cellulose, CMC (Sigma-Aldrich, catalog number: C5678 )
  37. Sterile deionized water (dH2O)
  38. Sterile double distilled water (ddH2O)
  39. Phenol (Fisher Scientific, catalog number: BP226500 )
  40. Chloroform (Fisher Scientific, catalog number: C607-4 )
  41. Isopropanol (Fisher Scientific, catalog number: A451SK-4 )
  42. Ethanol (Fisher Scientific, catalog number: A995-4 )
  43. Primers (Listed below in Table 1)
  44. dNTPs (Sigma-Aldrich, catalog number: DNTP100A-1KT )
  45. Polymerase for PCR (Fisher Scientific, catalog number: FB-6000-10 )
  46. iTaq Univeral SYBR Green Supermix (Bio-Rad, catalog number: 1725122 )
  47. Agarose (Fisher Scientific, catalog number: BP1356 )
  48. Soil mix (Fafard, catalog number: Fafard Growing Mix 2/C-2 )
  49. Peters 20:20:20 General Purpose fertilizer (Hummert International; catalog number: 07-5400-1 )
  50. F. graminearum macroconidia suspension (see Procedure)
  51. F. graminearum mycelial fragments (see Procedure)
  52. Potato Dextrose Agar-Half strength (½ PDA) (see Recipes)
  53. Carboxymethyl Cellulose (CMC) media (see Recipes)
  54. Arabidopsis DNA extraction buffer (see Recipes)
  55. Tris-equilibrated phenol-chloroform (see Recipes)
  56. Spray spore suspension (see Recipes)

Equipment

  1. Hand-held atomizer
  2. Micropipettes (P20, P100 and P1000)
  3. Standard Lab Incubator for cultivating fungus (Fisher Scientific, Fisher ScientificTM IsotempTM)
  4. Plant growth chamber for cultivating Arabidopsis (Percival scientific, model: AR-66L2 )
  5. Thermal cycler (Techne, model: 3PrimeX )
  6. Real-time PCR system (Ilumina, EcoTM, catalog number: EC-101-1001 )
    Note: This product has been discontinued by the manufacturer.
  7. Compound microscope (Leica, model: DM2000 )
  8. Tabletop centrifuge (Beckman)
  9. Microfuge (Fisher Scientific, Fisher ScientificTM accuSpinTM, model: Micro 17/Micro 17R )
  10. Vortex-Genie 2 (Scientific Industries, catalog number: SI-0236 )
  11. Basic power supply gel electrophoresis powerpack, trays and combs, (Bio-Rad, PowerPacTM, catalog number: 1645050 )
  12. Gel electrophoresis system (Bio-Rad, catalog number: 1704405 )

Software

  1. Variance (ANOVA) (P < 0.05) (SAS Institute Inc, SAS v5.1)

Procedure

Experiments in our lab have shown that placing F. graminearum mycelia or macroconidia on the leaf surface does not yield uniform infection. Further, the level of infection is also highly variable. However, leaves when infiltrated with fungal mycelium fragments, which are small enough to enter the leaf tissue presumably through stomatal openings, resulted in reproducible infection. Macroconidia on the other hand are larger and could not be easily infiltrated into the leaves. On Arabidopsis floral tissue, macroconidia are able to germinate and successfully infect tissue.

  1. Cultivation of Fusarium graminearum isolate Z-3639 and preparation of fungal mycelial suspension for inoculation of Arabidopsis leaves
    1. The fungus is cultivated and maintained on ½ strength Potato Dextrose Agar (PDA) made with 0.7% Agar. Plates are 100 mm (wide) x 15 mm (deep).
    2. To prepare fungi for inoculation, culture the F. graminearum isolate Z-3639 (Bowden and Leslie, 1999) on ½ PDA plates for 8-10 days at 28 °C. As the fungal mass grows, it turns a pinkish red color (Figure 1).
      Notes:
      1. If a 28 °C incubator is not available the fungus can be cultivated at room temperature, although preferably not below 22 °C.
      2. Plates with fungal mass that are older than 1 month should not be used in the preparation of inoculum.


      Figure 1. PDA plates showing red coloration due to Fusarium graminearum growth for 8 days at 28 °C

    3. After 10 days, flood each plate with 10 ml of sterile ddH2O, and carefully scrape mycelia from plate surface with a soft camel hair brush taking care not to scrape off the media (see Figure 2). This process harvests fungal mycelia from the media and simultaneously fragments it into smaller pieces, which is critical for the subsequent infection of Arabidopsis leaves. The fungal suspension will have a pink color to it (see Figure 2).
      Note: For control inoculations, use ½ PDA plates that were not inoculated with F. graminearum.
    4. The fungal suspension is filtered through four layers of cheesecloth (alternatively can use two layers of miracloth) to remove debris and larger mycelial mass (see Figure 2). Finally, after suspensions from 6-7 plates are collected, pass 5 ml of sterile ddH2O through the cheesecloth (or miracloth). Repeat this wash an additional time. Typically around 50 ml of suspension is required to infiltrate 60-70 leaves.


      Figure 2. Preparation of fungal mycelial fragments from ½ PDA plates. Left panel: Harvesting fungal mycelia from ½ PDA plates with a camel hair brush. Middle panel: Filtering fungal mycelial suspension through cheesecloth. Right panel: Culture tube with filtered fungal mycelial suspension.

  2. Cultivation of Fusarium graminearum isolate Z-3639 and preparation of fungal spores for inoculation of Arabidopsis floral tissues 
    1. Cultivate F. graminearum on ½ PDA plates for 8-10 days at 28 °C as described above.
    2. To promote sporulation, a 1/4th square inch of fungal plug of fungal mycelial mass is cut from the PDA plate that shows profuse fungal growth and placed in a 1 L conical flask containing 250 ml of sterile carboxymethyl cellulose media.
    3. Incubate the fungus-inoculated CMC media (see Recipes) containing flask on a shaker at 100 rpm at 28 °C for 7-9 days till profuse macroconidiation is observed.
    4. The fungal suspension is filtered through four layers of cheesecloth to remove debris and mycelial mass.
    5. The filtrate containing macroconidia is centrifuged in a table top swing-bucket centrifuge at 3,000 x g for 10 min.
    6. The pelleted macroconidia are washed by re-suspending them in 10 ml sterile ddH2O followed by centrifugation at 3,000 x g for 10 min at room temperature, as described above. This wash is repeated one more time.
    7. The pelleted macroconidia (Figure 3) is re-suspended to a concentration of 1 x 105 macroconidia/ml in sterile ddH2O water containing 0.001% Silwet L-77.


      Figure 3. Fusarium graminearum macroconidia. Bar represents 20 μm.

  3. Arabidopsis cultivation
    1. A compost-peat-based Fafard #2 soil mix was used for cultivating Arabidopsis. The soil was first sterilized by autoclaving as follows: Soil sufficient to half-fill an autoclave bag is mixed with sufficient water to until complete saturation. At the same time, care must be taken to break large clumps of soil to ensure uniform soil saturation.
    2. The loosely closed bag is autoclaved for 1 h. The soil is then allowed to cool to room temperature (overnight) before use.
    3. The autoclaved, but cooled soil was loosely packed into Kord brand 3.5” square pots with bottom holes that were placed in 20-9/16” x 10-3/16” x 2-3/8” 1020 tray with bottom holes, which were further placed in 20-9/16” x 10-3/16” x 2-3/8” 1020 tray without holes (see Figure 4).


      Figure 4. Arabidopsis cultivation set-up consisting of 3.5” pots contained in a tray with holes, which in turn is contained in a tray without holes

    4. The soil was sub-irrigated by filling the outermost tray with tap water containing at 0.4 g/gallon of Peters 20:20:20 fertilizer and placing the soil-filled pots contained in the tray with bottom holes in it.
    5. The soil was allowed to wet by capillary action till the soil surface was well wetted.
    6. Excess water was drained by lifting the tray with bottom holes containing the pots above the water level.
    7. The water in the flat without holes was drained off.
    8. The drained pots in the flat with bottom holes were returned to the tray without holes.
    9. Two seeds per pot were placed on the surface of the soil with a moistened toothpick, one seed at a time.
    10. After all pots were seeded, the entire set up of pots in trays was covered with a transparent DOM1020 plastic dome and transferred into a cold room where they were left in the dark for stratification.
    11. Two days later, the trays with the pots were moved into a growth room or growth chamber set at 22 °C under a 14 h light (80 μE m-2 sec-1)/10 h dark regime with approximately 60% relative humidity (RH).
    12. Approximately four-week-old plants were used for inoculating leaf tissue with fungal mycelial fragments, while 6-7 week old plants with unbranched bolts were used for inoculating the inflorescence tissue with fungal macroconidia.

  4. Arabidopsis leaf infection with Fusarium graminearum
    1. Inoculation of Arabidopsis leaves with Fusarium graminearum mycelial fragments
      1. Approximately four-week-old Arabidopsis plants were used. It is important to include the appropriate control genotypes in each experiment. Plants are watered the day before inoculation. Infection is typically done in the afternoon hours.
      2. Expanded leaves for inoculation are marked with a water-proof marker. Approximately 4-5 leaves per plant are inoculated. A minimum of 60 leaves from 15 plants of each genotype are required for each treatment (mock v/s fungus).
      3. A 1 ml needle-less syringe is used for infiltrating a suspension of fungal mycelial fragments into the abaxial side (underside) of the Arabidopsis leaves (Figure 5). Leaves are infiltrated on each side of the mid-vein till the entire leaf area is infiltrated. Control (mock) treatment involves water that was passed over the PDA plates without the fungus and processed similar to the processing of the fungal culture.
        Note: Use gloves, eye protection and lab coats when carrying out fungal inoculations. All waste coming in contact with the fungal culture is collected and autoclaved before disposing.
      4. After infiltration, plants are covered with a transparent dome for 48 h to maintain high humidity and promote fungal infection.


      Figure 5. Fungal infiltration into Arabidopsis leaves. Shown is fungal culture being infiltrated with a needle-less syringe into the abaxial surface (undersurface) of an Arabidopsis leaf.

    2. Scoring the severity of Fusarium graminearum disease on Arabidopsis leaves
      1. Disease spread is seen in the leaves as a spread of chlorosis and severity is recorded 5 days post inoculation. However, since disease progression depends on the quality of the fungal inoculum, if disease progression is slow then disease severity can be monitored at day 6 or even day 7.
      2. The percentage of inoculated leaves exhibiting chlorosis covering < 25% (category I), 25-50% (category II), 50-75% (category III) and > 75% (category IV) of leaf area are determined for each genotype (Figure 6). PCR analysis for fungal DNA relative to plant DNA is used to confirm fungal growth over the course of infection (Figure 7 left panel) and determine correlation between disease severity and fungal growth in the diseased leaves (Figure 7 right panel).
      3. A minimum of 60 leaves from 15 plants of each genotype are evaluated for disease severity.
      4. Disease severity index is calculated using the formula

        Where,
        I = disease severity score: 1 for category I, 2 for category II, 3 for category III and 4 for category IV,
        nI = number of leaves with each score,
        N = total number of leaves,
        k = highest score (in this case it is 4).
      5. Single factor analysis of variance (ANOVA) (P < 0.05) (SAS v5.1) is used to compare disease severity amongst different genotypes. Figure 8 shows a representative data set comparing the disease severity between wild type (WT) plants of Arabidopsis accession Columbia (Col-0) and two mutants, mut-1 and mut-2, that exhibit enhanced resistance.


      Figure 6. Disease symptoms in Fusarium graminearum-infected leaves. Diseased leaves are categorized into four groups based on the extent of leaf area exhibiting chlorosis.


      Figure 7. PCR analysis for fungal DNA. Left panel: PCR (25 and 30 cycles) for fungal and plant genes on DNA extracted from infected leaves exhibiting < 25% (lane 1), 25-50% (lane 2), 50-75% (lane 3), and > 75% (lane 4) chlorosis. Right panel: PCR (25 and 30 cycles) for fungal and plant genes on DNA extracted from 15 pooled leaves collected at 2, 4 and 6 days post inoculation (dpi). PCR was conducted with primers specific for the fungal NahG (FgNahG) and TUBULIN (FgTUB) genes, and as a control the Arabidopsis ACT8 (AtACT8) gene.


      Figure 8. Fusarium graminearum disease severity in leaves of Arabidopsis wild-type accession Col-0 and two mutants (mut-1 and mut-2) that exhibit reduced disease severity. The percentage of inoculated leaves exhibiting chlorosis covering < 25%, 25-50%, 50-75% and > 75% of the leaf area at 5 dpi was determined for each genotype. The disease severity index for each genotype is indicated on the right.

    3. Monitoring fungal growth by PCR
      Infected leaf tissue is processed to extract DNA, which includes both, plant and fungal DNA.
      1. DNA extraction
        1. Fifteen fungus-inoculated and as control mock-inoculated leaves per Arabidopsis genotype are randomly harvested, pooled and quick frozen in liquid nitrogen.
        2. The frozen tissue is ground to a powder in a chilled mortar with a chilled pestle. Approximately 50 mg of the frozen powder is transferred into a 1.7 ml microfuge tube containing 200 μl of DNA extraction buffer (see Recipes) at room temperature and mixed and left at room temperature for a minimum of 5 min to allow tissue dissociation.
        3. Once all samples are processed, they are placed in a microfuge and centrifuged at 16,500 x g for 5 min to pellet cell debris.
        4. The supernatant is transferred into a fresh 1.7 ml microfuge tube and mixed with 100 μl of Tris-equilibrated phenol-chloroform (pH 7-8) (see Recipes). Samples are centrifuged at 16,500 x g for 10 min at room temperature in a microfuge.
        5. The DNA containing supernatant is transferred into a fresh tube containing 150 μl of isopropanol. Vortex for 5 sec and leave samples at room temperature for 10 min.
        6. Pellet DNA by centrifugation at 16,500 x g for 10 min at room temperature. Wash pellet with 500 μl 70% ethanol and then let pellet air dry left inverted on kimwipes for 10 min.
        7. Dissolve the pellet, which contains a mix of plant and fungal DNA in 200 μl of ddH2O.
      2. PCR for monitoring fungal growth in Arabidopsis leaves
        1. Primers designed to the fungal genes FgNahG and FgTUB are used to monitor the amount of fungus relative to Arabidopsis ACT8.
        2. 1 μl of DNA extracted from plant tissue is used for PCR in a total 20 μl volume containing 0.25 μM each of dATP, dTTP, dCTP and dGTP, 0.05 μM of each primer, and 1 unit of Taq Pol (or related polymerase) along with the appropriate PCR buffer.
        3. PCR was conducted using the following amplification protocol: 3 min at 94 °C for denaturation of nucleic acids, followed by 25 or 30 cycles of 94 °C for 30 sec, 58 °C for 30 sec and 72 °C for 30 sec, culminating with a step at 72 °C for 30 sec and a hold step at 4 °C.
        4. The PCR products were resolved on 1.5% agarose gel, stained with ethidium bromide and visualized under UV illumination (see Figure 7 as an example).
      3. qPCR-based quantification of fungal growth in Arabidopsis leaves
        Quantitative PCR (qPCR) for fungal genes (FgNahG and FgTUB) was performed with Sybr® Green PCR Master Mix on an Eco Illumina system (or any comparable real-time PCR machine) using the following amplification protocol: 10 min at 95 °C for polymerase activation and denaturation of nucleic acids, followed by 40 cycles of 95 °C for 10 sec, 58 °C for 30 sec and 72 °C for 30 sec. This was followed by a product melt to confirm a single PCR product. The level of gene expression was normalized to that of Arabidopsis EF1α by subtracting the CT value of EF1α from the CT value for the fungal gene. The ΔΔCT method (Livak and Schmittgen, 2001) was used to calculate relative fold changes. See Figure 9 as an example.


        Figure 9. qPCR analysis to monitor fungal growth. qPCR analysis of fungal FgNahG gene relative to that of Arabidopsis EF1α conducted on DNA extracted from Fg-infected leaves at 4 and 6 dpi.

        Table 1. PCR primers


  5. Disease evaluation of Arabidopsis inflorescence tissue infected with Fusarium graminearum
    1. Plant Inoculation
      1. Select flowering plants for inoculation i.e., choose plants that possess an unbranched bolt with both open flowers on the terminal inflorescence and two to three developing siliques. It is important to include the appropriate control genotypes with each experiment. 
        Note: A minimum of ten plants should be chosen for inoculation. A similar number of control plants are sprayed with water.
      2. With a permanent black marker, Mark the position on the flower stem above which only open flowers are present and below which siliques have begun developing.
        Note: In order to minimize experimental error set up the plants in a randomized block design.
      3. Spray spore suspension (1 x 105 spores/ml in sterile ddH2O water containing 0.001% Silwet L-77) using a hand-held atomizer until droplet run-off has commenced.
      4. After this, re-inoculate each inflorescence with inoculum dispensed from the same sprayer (four sprays per flower head).
      5. Control plants are inoculated in the same way using de-ionized water containing 0.001% Silwet L-77.
      6. The inoculated plants were covered with a transparent plastic bag to ensure high humidity and placed in a plant growth chamber set at 22 °C under a 14 h light (80-100 μE m-2 sec-1)/10 h dark regime. Three days later the plastic bag was removed and the plants left in the growth chamber for an additional 4 days.
    2. Fusarium-Arabidopsis Disease (FAD) score
      1. Disease symptoms on individual inflorescence were monitored from day 3 onwards with the final disease score taken at 7 days post inoculation. However, if the progression of disease is rapid then the final disease score can be taken on day 5 or 6.
      2. A numerical scoring system developed by Urban et al. (2002) (Table 2) was used to obtain the Fusarium-Arabidopsis Disease (FAD) score.
      3. Disease phenotypes are assessed for three separate floral subcomponents (i) Flowers (F): infection covering open flowers and buds. (ii) New siliques (NS): Infection severity on siliques that developed after inoculation from flowers that were fully open at the time of inoculation. These flowers were located above the permanent mark placed on the stem at the time of fungal inoculation. (iii) Older siliques (OS): infection severity on siliques that existed at the time of fungal inoculation. For each of these components, the severity of infection is classified based on the macroscopic assessment of symptoms (Figure 10), as denoted in Table 2.
      4. The final Fusarium-Arabidopsis disease (FAD) value is calculated by addition of the three subcomponent scores, i.e., F + NS + OS = FAD as described by Urban et al. (2002). A representative data set is presented in Table 3.
      5. Arabidopsis genotypes with FAD values of 3 and below are classified as exhibiting resistance to Fg whereas those with values of 10 and above are classified as susceptible.

      Table 2. Classification of disease phenotypes and scores for flower, and new and old siliques. Adapted from Urban et al. (2002).

      aSiliques formed during the seven day period after fungal inoculation.
      bSiliques that were present when plants were sprayed with fungal macroconidia


      Figure 10. Symptoms of Fusarium graminearum disease in Arabidopsis inflorescence. A. A susceptible inflorescence showing disease symptoms. B. A relatively resistant inflorescence showing production of new flowers and siliques developed from flowers that were present seven days earlier at the time of fungal macroconidia inoculation.

      Table 3. Fusarium graminearum disease on inflorescence of Arabidopsis WT accession Col-0 and the mut-1 and mut-2 mutants

Recipes

  1. Potato Dextrose Agar-Half strength (½ PDA)
    Potato dextrose broth powder 19.5 g
    Agar 7.0 g
    Distilled Water 1,000 ml
    Adjust pH to 5.6 ± 0.2 at 25 °C, prior to adding Agar.
    Sterilize by autoclaving for 20 min. Pour and allow to set approximately 30 ml into each 100 x 15 mm petri dish.
  2. Carboxymethyl Cellulose (CMC) media
    NH4NO3 1.0 g
    KCl 0.2 g
    MgSO4·7H2O 1.0 g
    Yeast extract 1.0 g
    Carboxymethyl cellulose 26.0 g
    Distilled water to 1,000 ml
    Sterilize by autoclaving for 20 min.
  3. Arabidopsis DNA extraction buffer
    200 mM Tris-Cl, pH 7.5 (Sambrook et al., 1989)
    250 mM NaCl
    25 mM EDTA, pH 7.5 (Sambrook et al., 1989)
    0.5% SDS
  4. Tris-equilibrated phenol-chloroform (Sambrook et al., 1989)
  5. Spray spore suspension
    1 x 105 spores/ml in sterile ddH2O water containing 0.001% Silwet L-77

Acknowledgments

This work was supported by funding from: the U.S. Department of Agriculture (Agreement #59-0200-3-003 and 59-0790-8-060) as cooperative projects with the U.S. Wheat & Barley Scab Initiative. The protocols described here are based on the procedures developed and described by Makandar et al. (2010), Nalam et al. (2015) and Urban et al. (2002).

References

  1. Bowden, R. L. and Leslie, J. F. (1999). Sexual Recombination in Gibberella zeae. Phytopathology 89(2): 182-188.
  2. Livak, K. J. and Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25(4): 402-408.
  3. Makandar, R., Nalam, V., Chaturvedi, R., Jeannotte, R., Sparks, A. A. and Shah, J. (2010). Involvement of salicylate and jasmonate signaling pathways in Arabidopsis interaction with Fusarium graminearum. Mol Plant Microbe Interact 23(7): 861-870.
  4. Nalam, V. J., Alam, S., Keereetaweep, J., Venables, B., Burdan, D., Lee, H., Trick, H. N., Sarowar, S., Makandar, R. and Shah, J. (2015). Facilitation of Fusarium graminearum infection by 9-Lipoxygenases in Arabidopsis and Wheat. Mol Plant Microbe Interact 28(10): 1142-1152.
  5. Sambrook, J., Fritch, E. F. and Maniatis, T. (1989). Molecular Cloning: a laboratory manual. Cold Spring Harbor Laboratory.
  6. Urban, M., Daniels, S., Mott, E., and Hammond-Kosack, K. (2002). Arabidopsis is susceptible to the cereal ear blight fungal pathogens Fusarium graminearum and Fusarium culmorum. Plant J 32: 961-973.

简介

禾谷镰刀菌(Fg)是小麦(小麦),燕麦( Avena sativa )和大麦镰刀菌 ),其针对花组织,从而不利地影响谷物产量和质量。由F生产的霉菌毒素。禾谷镰菌进一步限制了感染谷物的消耗性。在实验室,F。禾谷镰刀菌也具有定居拟南芥的叶和花序组织的能力。 A之间的交互。 thaliana 和 F。禾谷镰刀菌(graminearum)提供了大量遗传和分子工具来研究植物和真菌之间的相互作用。禾本科(Graminearum)来阐明植物基因和促进抗性的途径,以及研究真菌如何靶向植物基因和促进疾病的机制。下面描述的方法允许有效地感染拟南芥叶和花序,以及评价疾病进展和真菌生长。拟南芥中的病害传播可以通过叶组织的萎黄病和花序组织的包括真菌团块在花序组织表面上的病害表型的视觉观察来容易地监测。可以通过聚合酶链反应(PCR)和定量实时PCR(qPCR)测量宿主组织中的Fg DNA的相对量来进一步监测真菌生长。

材料和试剂

  1. PCR管(Fisher Scientific,目录号:14222262)
  2. 培养皿(100×15mm)(Fisher Scientific,目录号:FB0875713)
  3. 50ml塑料螺旋管(Midsci,目录号:C50B)
  4. 移液管吸头(无菌)(Midsci,目录号:AVR-1,AVR-4和AVR-11)
  5. 1.7ml微量离心管(无菌)(目录号:AVSS1700)
  6. 来自当地工艺品店或Miracloth(EMD Millipore,目录号:475855-1R)的Cheesecloth
  7. 文化管
  8. 1ml无针注射器(Tuberculin注射器)(Becton Dickinson,目录号:309659)
  9. 漏斗
  10. 1L玻璃锥形瓶(Pyrex牌)
  11. 镊子
  12. 血细胞计数器
  13. 骆驼毛刷
  14. Sharpie或类似的防水标记
  15. 一次性手套
  16. Kimwipes,棉纸或纸巾
  17. 面罩(Fisher Scientific,目录号:18-999-4542)
  18. Kord品牌3.5英寸方底盆底部孔(Hummert International,目录号:12-1350-1)
  19. 至。具有底孔的塑料标准平台1020托盘(Hummert International,目录号:11-3000-1)
  20. 至。 Plastics Standard Flats 1020 tray without holes(Hummert International,catalog number:11-3050-1)
  21. DOM1020塑料圆顶适合1020单位(悍马国际,目录号:11-3360-1)
  22. 透明塑料袋(高达13加仑回收束带清除垃圾袋)
  23. 禾本科镰孢菌分离Z-3639(Bowden和Leslie,1999)
  24. 拟南芥种子(Accession Columbia,N?ssen,and Wassilewskija)
  25. Silwet L-77(Lehle种子,目录号:VIS-30)
  26. 马铃薯葡萄糖肉汤(Becton Dickinson,目录号:254920)
  27. 酵母提取物(Becton Dickinson,目录号:212750)
  28. BD Difco琼脂(Becton Dickinson,目录号:214530)
  29. 硝酸铵(Fisher Scientific,目录号:A676)
  30. 氯化钾(Fisher Scientific,目录号:P217)
  31. 硫酸镁七水合物(Fisher Scientific,目录号:M63)
  32. 氯化钠(Fisher Scientific,目录号:BP358-1)
  33. Tris-Base(Fisher Scientific,目录号:BP152)
  34. 乙二胺四乙酸二钠盐二水合物(Fisher Scientific,目录号:S311)
  35. 十二烷基硫酸钠(Fisher Scientific,目录号:BP166)
  36. 羧甲基纤维素CMC(Sigma-Aldrich,目录号:C5678)
  37. 无菌去离子水(dH 2 O)
  38. 无菌双蒸水(ddH 2 O)
  39. 苯酚(Fisher Scientific,目录号:BP226500)
  40. 氯仿(Fisher Scientific,目录号:C607-4)
  41. 异丙醇(Fisher Scientific,目录号:A451SK-4)
  42. 乙醇(Fisher Scientific,目录号:A995-4)
  43. 引物(列于下表1)
  44. dNTP(Sigma-Aldrich,目录号:DNTP100A-1KT)
  45. PCR用聚合酶(Fisher Scientific,目录号:FB-6000-10)
  46. iTaq Univeral SYBR Green Supermix(Bio-Rad,目录号:1725122)
  47. 琼脂糖(Fisher Scientific,目录号:BP1356)
  48. 土壤混合物(Fafard,目录号:Fafard Growing Mix 2/C-2)
  49. Peters 20:20:20通用化肥(Hummert International;目录号:07-5400-1)
  50. F。禾谷镰菌大宏观悬浮液(见程序)
  51. F。禾谷镰菌菌丝体碎片(见程序)
  52. 马铃薯葡萄糖琼脂 - 半强度(?PDA)(见配方)
  53. 羧甲基纤维素(CMC)培养基(参见配方)
  54. 拟南芥 DNA提取缓冲液(参见配方)
  55. 三平衡苯酚 - 氯仿(见配方)
  56. 喷雾孢子悬浮液(见配方)

设备

  1. 手持式雾化器
  2. 微量移液器(P20,P100和P1000)
  3. 用于培养真菌的标准实验室培养箱(Fisher Scientific,Fisher Scientific TM Isotemp TM
  4. 用于培养拟南芥的植物生长室(Percival scientific,型号:AR-66L2)
  5. 热循环仪(Techne,型号: 3 PrimeX)
  6. 实时PCR系统(Ilumina,Eco TM ,目录号:EC-101-1001)
    注意:此产品已由制造商停用。
  7. 复合显微镜(Leica,型号:DM2000)
  8. 台式离心机(Beckman)
  9. Microfuge(Fisher Scientific,Fisher Scientific accuSpin TM ,型号:Micro 17/Micro 17R)
  10. Vortex-Genie 2(Scientific Industries,目录号:SI-0236)
  11. 基本电源凝胶电泳电源组,托盘和梳子(Bio-Rad,PowerPac TM ,目录号:1645050)
  12. 凝胶电泳系统(Bio-Rad,目录号:1704405)

软件

  1. 方差(ANOVA)(
    <0.05)(SAS Institute Inc,SAS v5.1)

程序

我们实验室的实验表明,放置 F。禾谷镰孢菌的菌丝体或大叶子在叶片表面不产生均匀感染。此外,感染的水平也是高度可变的。然而,当浸润有真菌菌丝体片段的叶子,其足够小以致可能通过气孔开口进入叶组织,导致可再现的感染。另一方面,大分生孢子较大,不能容易地渗入叶中。在拟南芥花卉组织上,大分生孢子能够发芽并成功感染组织。

  1. 禾谷镰孢分离株Z-3639的培养和用于接种拟南芥叶的真菌菌丝体悬浮液的制备
    1. 将真菌培养并保持在用0.7%琼脂制成的1/2强度马铃薯葡萄糖琼脂(PDA)上。平板为100 mm(宽)x 15 mm(深)。
    2. 为了制备真菌用于接种,培养真菌。禾本科(Graminearum)在?PDA平板上在28℃下分离Z-3639(Bowden和Leslie,1999)8-10天。随着真菌生长,它变成粉红色的红色(图1)。
      注意:
      1. 如果不存在28℃的培养箱,则真菌可以在室温下培养,但优选不低于22℃。
      2. 具有大于1个月的真菌质量的板不应用于接种物的制备。


      图1.在28℃下由于禾本科禾谷镰孢生长8天而显示红色的PDA平板

    3. 10天后,用10ml无菌ddH 2 O淹没每个平板,并用软骆驼毛刷小心地从平板表面刮下菌丝体,注意不要刮去介质(参见图2)。该方法从培养基中收获真菌菌丝体,并同时将其分成较小的片段,这对随后的拟南芥叶的感染是关键的。真菌悬浮液将具有粉红色的颜色(见图2)。
      注意:对于对照接种,使用未接种禾谷镰孢的1/2 PDA平板。
    4. 真菌悬浮液通过四层粗纱布过滤(或者可以使用两层miracloth)以除去碎片和较大的菌丝团(见图2)。最后,在收集来自6-7板的悬浮液后,使5ml无菌ddH 2 O通过粗棉布(或miracloth)。重复此洗涤额外的时间。通常需要约50ml的悬浮液来渗透60-70片叶子

      图2.从?PDA平板制备真菌菌丝体片段。左图:用骆驼毛刷从?PDA平板收获真菌菌丝体。中图:通过干酪包布过滤真菌菌丝体悬浮液。右图:具有过滤的真菌菌丝体悬浮液的培养管
  2. 禾本科镰孢菌的分离分离Z-3639和制备用于接种拟南芥花卉组织的真菌孢子
    1. 培养 F。禾谷镰孢在?PDA平板上在28℃下培养8-10天,如上所述。
    2. 为了促进孢子形成,从显示出大量真菌生长的PDA平板上切下真菌菌丝体团的1/4英寸平方英寸的真菌塞,并放置在含有250ml无菌羧甲基的1L锥形瓶中纤维素介质
    3. 将真菌接种的CMC培养基(参见Recipes)在摇瓶上以100rpm在28℃下培养7-9天,直到观察到大量泛素化。
    4. 真菌悬浮液通过四层干酪布过滤以除去碎片和菌丝团。
    5. 将含有大分子的滤液在台式摇摆式离心机中以3,000xg离心10分钟。
    6. 通过将沉淀的大分生孢子重悬浮于10ml无菌ddH 2 O中,然后如上所述在室温下以3,000xg离心10分钟来洗涤沉淀的大分生孢子。该洗涤再重复一次。
    7. 将沉淀的大分生孢子(图3)在含有0.001%Silwet L-77的无菌ddH 2 O水中再悬浮至1×10 5大分生孢子/ml的浓度。


      图3. 禾本科镰刀菌大分生孢子。条形代表20μm。

  3. 拟南芥栽培
    1. 使用基于堆肥的Fafard#2土壤混合物培养拟南芥。土壤首先通过高压灭菌如下灭菌:将足以半高压灭菌高压釜袋的土壤与足够的水混合直至完全饱和。同时,必须小心打破大块土壤以确保均匀的土壤饱和。
    2. 将松散封闭的袋高压灭菌1小时。然后使土壤冷却至室温(过夜),然后使用。
    3. 将经高压灭菌,但冷却的土壤松散地装入具有底部孔的Kord品牌3.5"方形罐中,所述底部孔放置在具有底部孔的20-9/16"×10-3/16"×2-3/8"进一步放置在没有孔的20-9/16"×10-3/16"×2-3/8"1020盘中(参见图4)。


      图4. 拟南芥栽培装置,其包含在具有孔的托盘中的3.5"盆,其又包含在没有孔的托盘中 >

    4. 通过用含有0.4g /加仑的Peters 20:20:20肥料的自来水填充最外层托盘并将放置在其中具有底孔的托盘中的填充土壤的盆放入其中来对土壤进行灌溉。
    5. 通过毛细作用使土壤湿润,直到土壤表面润湿良好。
    6. 通过提起具有底部孔的托盘排出多余的水,该底部孔包含在水位以上的罐。
    7. 没有孔的平面中的水被排出。
    8. 将带有底孔的平板中的排水罐返回到没有孔的托盘。
    9. 每盆两个种子用润湿的牙签放在土壤表面,每次一个种子。
    10. 在所有盆播种后,用透明DOM1020塑料圆顶覆盖托盘中的整个盆设置,并转移到冷室中,在那里将其放置在黑暗中用于分层。
    11. 两天后,将具有罐的托盘移至设定在22℃的生长室或生长室中,在14小时光照(80μEm -2s -1 <1s -1)下)/10h黑暗区域,具有约60%的相对湿度(RH)。
    12. 大约四周龄的植物用于用真菌菌丝体片段接种叶组织,而具有无分支螺栓的6-7周龄植物用于用真菌大分生孢子接种花序组织。

  4. 禾本科镰刀菌 叶感染
    1. 用禾本科禾谷镰孢菌菌体碎片接种拟南芥叶
      1. 使用约四周龄的拟南芥植物。在每个实验中包括适当的对照基因型是重要的。在接种前一天浇水植物。感染通常在下午时间完成。
      2. 用于接种的扩展叶用防水标记标记。每株植物接种约4-5片叶。每种处理需要来自每种基因型的15株植物的至少60片叶子(模拟v/s真菌)。
      3. 使用1ml无针注射器将真菌菌丝体碎片的悬浮液渗入拟南芥叶的背面(下侧)(图5)。叶浸润在中间静脉的每一侧,直到整个叶区域浸润。对照(模拟)处理涉及通过没有真菌的PDA平板上的水,并且类似于真菌培养物的处理。
        注意:进行真菌接种时,请使用手套,眼睛保护和实验室外套。收集所有与真菌培养物接触的废物,并在处理前进行高压灭菌。
      4. 浸润后,用透明圆顶覆盖植物48小时以保持高湿度并促进真菌感染。


      图5.真菌浸润到拟南芥叶中。显示的是用无针注射器将真菌培养物浸润到拟南芥叶的背面(下表面) em>叶。

    2. 对拟南芥叶片禾谷镰孢菌病的严重程度进行评分
      1. 在接种后5天,在叶中观察到病害传播,作为褪绿的扩展和严重性。然而,由于疾病进展取决于真菌接种物的质量,如果疾病进展缓慢,则可以在第6天或甚至第7天监测疾病严重性。
      2. 接种的叶子的百分比, 25%(I类),25-50%(II类),50-75%(III类)和>对于每种基因型确定75%(IV类)叶面积(图6)。对于相对于植物DNA的真菌DNA的PCR分析用于确认感染过程中的真菌生长(图7左图),并确定疾病严重性和病叶中真菌生长之间的相关性(图7右图)。
      3. 评估来自每种基因型的15株植物的最少60片叶的疾病严重性。
      4. 使用公式
        计算疾病严重程度指数
        在哪里,
        I =疾病严重程度分数:1类,I类,2类,II类,3类,III类,4类,IV类 n I =每个分数的叶数,
        N =总叶数,
        k =最高分(在这种情况下为4分)。
      5. 使用单因素方差分析(ANOVA)(P <0.05)(SAS v5.1)来比较不同基因型中的疾病严重性。图8显示了比较拟南芥登记号Columbia(Col-0)的野生型(WT)植物与表现出增强抗性的两种突变体mut-1和mut-2之间的疾病严重性的代表性数据集。


      图6.在禾本科镰刀菌感染的叶中的疾病症状。根据显示缺绿的叶面积的程度,将疾病叶分为四组。


      图7.真菌DNA的PCR分析。左图:对从感染叶提取的DNA上的真菌和植物基因进行PCR(25和30个循环) 25%(泳道1),25-50%(泳道2),50-75%(泳道3),和> 75%(泳道4)萎黄病。右图:从接种后第2,4和6天(dpi)收集的15个合并叶中提取的DNA上的真菌和植物基因的PCR(25和30个循环)。用对真菌NahG( FgNahG )和 TUBULIN ( FgTUB )基因特异性的引物进行PCR, a控制拟南芥ACT8 ( AtACT8 )基因。

      图8.拟南芥野生型登录Col-0和两个突变体(mut-1和mut-2)叶片中的禾谷镰孢菌病病情严重性接种的叶的百分比, 25%,25-50%,50-75%和>对于每种基因型确定5dpi处的叶面积的75%。每种基因型的疾病严重程度指数显示在右侧
    3. 通过PCR监测真菌生长
      处理感染的叶组织以提取DNA,其包括植物和真菌DNA。
      1. DNA提取
        1. 随机收获15个接种真菌和作为对照的模拟接种的每个拟南芥基因型的叶子,在液氮中快速冷冻。
        2. 将冷冻的组织在具有冷却的研杵的冷冻研钵中研磨成粉末。在室温下将约50mg冷冻粉末转移到含有200μlDNA提取缓冲液(参见Recipes)的1.7ml微量离心管中并混合,并在室温下放置至少5分钟以允许组织解离。 >
        3. 一旦所有样品被处理,将它们置于微量离心机中并在16,500×g离心5分钟以沉淀细胞碎片。
        4. 将上清液转移到新鲜的1.7ml微量离心管中,并与100μlTris-平衡的酚 - 氯仿(pH7-8)(参见Recipes)混合。将样品在室温下在microfuge中以16,500×g离心10分钟。
        5. 将含有DNA的上清液转移到含有150μl异丙醇的新管中。涡旋5秒,并将样品在室温下放置10分钟
        6. 通过在室温下以16,500×g离心10分钟沉淀沉淀DNA。用500μl70%乙醇洗涤沉淀,然后让沉淀物空气干燥,在kimwipes上倒置10分钟
        7. 将含有植物和真菌DNA的混合物的沉淀溶解在200μlddH 2 O中。
      2. 用于监测拟南芥叶中真菌生长的PCR
        1. 针对真菌基因设计的引物fgNahG 和 FgTUB 用于监测相对于拟南芥ACT8的真菌数量。
        2. 将从植物组织提取的1μlDNA用于总共20μl体积的PCR,所述体积含有各0.25μM的dATP,dTTP,dCTP和dGTP,0.05μM的每种引物和1单位的Taq Pol(或相关聚合酶),以及合适的PCR缓冲液
        3. 使用以下扩增方案进行PCR:94℃3分钟,核酸变性,接着94℃30秒,58℃30秒和72℃30秒的25或30个循环,最终达到在72℃下步骤30秒,在4℃下保持步骤
        4. PCR产物在1.5%琼脂糖凝胶上分离,用溴化乙锭染色并在UV照射下可见(作为实例参见图7)。
      3. 基于qPCR的定量拟南芥叶中真菌生长
        在Eco Illumina系统上使用Sybr?Green PCR Master Mix进行真菌基因(fgNahG 和 FgTUB )的定量PCR(qPCR)任何可比较的实时PCR仪)使用以下扩增方案:在95℃下10分钟用于聚合酶活化和核酸变性,随后是95℃10秒,58℃30秒和72℃的40个循环30秒。随后是产物熔化以确认单一PCR产物。通过从C T中减去EF1α的C T值,将基因表达水平标准化为拟南芥EF1α的水平。真菌基因的亚基值。使用ΔΔC方法(Livak和Schmittgen,2001)计算相对倍数变化。以图9为例。


        图9.用于监测真菌生长的qPCR分析相对于拟南芥EF1α的真菌FgNahG 基因的qPCR分析对从提取的DNA进行的 > Fg - 感染的叶子。

        表1. PCR引物


      4. 通过用含有0.4g /加仑的Peters 20:20:20肥料的自来水填充最外层托盘并将放置在其中具有底孔的托盘中的填充土壤的盆放入其中来对土壤进行灌溉。
      5. 通过毛细作用使土壤湿润,直到土壤表面润湿良好。
      6. 通过提起具有底部孔的托盘排出多余的水,该底部孔包含在水位以上的罐。
      7. 没有孔的平面中的水被排出。
  5. 感染了禾本科禾谷镰菌的拟南芥花粉组织的疾病评价
    1. 植物接种
      1. 选择用于接种的开花植物,即选择具有在末端花序上具有开放花的无枝的螺栓和两至三个发育的长角果的植物。在每次实验中包括适当的对照基因型是重要的。
        注意:应选择至少10株植物进行接种。用水喷洒相似数量的对照植物。
      2. 有一个永久的黑色标记,标记在花茎上的位置,只有开放的花存在,在下面的长角果已经开始发展。
        注意:为了最小化实验误差,在随机区组设计中设置植物。
      3. 使用手持式雾化器喷雾孢子悬浮液(1×10 5孢子/ml,在含有0.001%Silwet L-77的无菌ddH 2 O水中)直至液滴滴落已开始。
      4. 之后,用同一喷雾器分配的接种物再次接种每个花序(每个花头四个喷雾)。
      5. 以相同的方式使用含有0.001%Silwet L-77的去离子水接种对照植物。
      6. 用透明塑料袋覆盖接种的植物以确保高湿度,并置于设定在22℃的植物生长室中,在14小时光照(80-100μEm -2 s -1, -1 )/10小时暗区。三天后,取出塑料袋,植物在生长室中再放置4天。
    2. 镰刀菌 - 拟南芥病疾病(FAD)评分
      1. 从第3天起监测个体花序的疾病症状,在接种后7天取得最终疾病评分。然而,如果疾病的进展快速,那么最终的疾病得分可以在第5或6天进行
      2. 由Urban等人开发的数字评分系统。 (2002)(表2)用于获得镰孢 - 拟南芥病(FAD)评分。
      3. 针对三个独立的花亚组分评估疾病表型(i)花(F):覆盖开放花和芽的感染。 (ii)新长角果(NS):在从接种时完全开放的花接种后形成的长角果上的感染严重性。这些花位于真菌接种时位于茎上的永久标记的上方。 (iii)较老的长角果(OS):在真菌接种时存在的长角果的感染严重性。对于这些组分中的每一种,基于症状的宏观评估(图10)对感染的严重性进行分类,如表2所示。
      4. 通过加入三个亚组分分数来计算最终的镰孢属 - 拟南芥病(FAD)值,即F + NS + OS = FAD,如Urban > et al 。 (2002)。代表性数据集在表3中给出。
      5. FAD值为3及以下的拟南芥基因型被分类为表现出对Fg的抗性,而具有10以上的值的那些基因型被分类为易感的。

      表2.疾病表型的分类以及花,新老角果的分数。改编自Urban等人。 (2002)。

      在真菌接种后七天期间形成的丝状体 b 在用真菌大分生孢子喷洒植物时存在的短枝

      图10.拟南芥花序中的禾本科禾谷镰孢病的症状。 A.显示疾病症状的敏感花序。 B.相对耐受的花序,显示由在真菌大分生孢子接种时7天前出现的花产生的新鲜花和长角果。

      表3.拟南芥 WT登录Col-0和mut-1和em-mut的花序上的禾谷镰孢菌病 2 突变体

食谱

  1. 马铃薯葡萄糖琼脂 - 半强度(?PDA)
    马铃薯葡萄糖肉汤粉末19.5克
    琼脂7.0克
    蒸馏水1000 ml
    在添加琼脂之前,在25℃下将pH调节至5.6±0.2 通过高压灭菌20分钟灭菌。倒入并允许在每个100×15mm培养皿中设置约30ml
  2. 羧甲基纤维素(CMC)培养基
    NH 4 NO 3 Sub 1.0g
    KCl 0.2 g
    MgSO 4·7H 2 O 1.0g
    酵母提取物1.0g
    羧甲基纤维素26.0克
    蒸馏水至1000 ml
    通过高压灭菌20分钟灭菌。
  3. 拟南芥 DNA提取缓冲区
    200mM Tris-Cl,pH 7.5(Sambrook等人,1989) 250mM NaCl 25mM EDTA,pH 7.5(Sambrook et al。,1989)
    0.5%SDS
  4. 三平衡的苯酚 - 氯仿(Sambrook等人,1989)
  5. 喷雾孢子悬浮液
    1×10 5孢子/ml,在含有0.001%Silwet L-77的无菌ddH 2 O水中。

致谢

这项工作得到了来自美国农业部(协议#59-0200-3-003和59-0790-8-060)的资助,作为与美国小麦&大麦痂病倡议。这里描述的方案基于由Makandar等人开发和描述的程序。 (2010),Nalam等人。 (2015)和Urban 等。 (2002)。

参考文献

  1. Bowden,RL和Leslie,JF(1999)。  性在玉米赤霉中重组。 Phytopathology 89(2):182-188。
  2. Livak,KJ和Schmittgen,TD(2001)。  分析的相对基因表达数据,使用实时定量PCR和2(-DΔDelta C(T))方法。方法 25(4):402-408。
  3. Makandar,R.,Nalam,V.,Chaturvedi,R.,Jeannotte,R.,Sparks,AA和Shah,J。(2010)。  简化禾本科镰刀菌 拟南芥和小麦中的9-脂氧合酶的感染。 Mol Plant Microbe Interact 28(10):1142-1152。
  4. Sambrook,J.,Fritch,EF和Maniatis,T。(1989)。  分子克隆:实验室手册 Cold Spring Harbor Laboratory 。
  5. Urban,M.,Daniels,S.,Mott,E.,and Hammond-Kosack,K.(2002)。  拟南芥易受谷物耳枯萎真菌病原体镰刀菌和禾谷镰孢菌的侵袭, em> Fusarium culmorum 。 32:961-973。

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Copyright: © 2016 The Authors; exclusive licensee Bio-protocol LLC.
引用:Nalam, V. J., Sarowar, S. and Shah, J. (2016). Establishment of a Fusarium graminearum Infection Model in Arabidopsis thaliana Leaves and Floral Tissues. Bio-protocol 6(14): e1877. DOI: 10.21769/BioProtoc.1877.
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