Mar 07, 2024
Arabidopsis thaliana detached leaf assay for the disease assessment of Fusarium graminearum infection
- Victoria J Armer1,2,
- Michael J. Deeks2,
- Kim E. Hammond-Kosack1
- 1Rothamsted Research;
- 2University of Exeter
Protocol Citation: Victoria J Armer, Michael J. Deeks, Kim E. Hammond-Kosack 2024. Arabidopsis thaliana detached leaf assay for the disease assessment of Fusarium graminearum infection. protocols.io https://dx.doi.org/10.17504/protocols.io.261gedmwov47/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 05, 2024
Last Modified: March 07, 2024
Protocol Integer ID: 94706
Keywords: Fusarium graminearum, Arabidopsis thaliana, detached leaf, Deoxynivalenol (DON)
Funders Acknowledgement:
UK BBSRC South West Biosciences Doctoral Training Partnership
Grant ID: BB/T008741/1
UK BBSRC Delivering Sustainable Wheat Grant (DSW)
Grant ID: BB/X011003/1
UK BBSRC Delivering Resilience to Biotic Stress
Grant ID: BBS/E/RH/230001B
UK BBSRC Designing Future Wheat
Grant ID: BB/P016855/1
Abstract
Arabidopsis thaliana (At), as the plant model organism, has the advantage of a bank of genotypes in stock centres worldwide. This allows for the assessment of genotypes and their contributes to disease resistance and/or susceptibility to a wide range of pathogens. We found that, through utilising the SALK lines (Alonso et al., 2003) provided by NASC (Nottingham, UK), to ensure reproducible disease phenotypes for disease analysis required additional specific steps not detailed in the current literature. This is likely due to the specificity of Fusarium graminearum (Fg) infection to the floral tissues of small grain cereals and the fact that At is a non-host of Fg. Whilst this protocol has been written and tested for the assessment of detached At leaves infected with Fg, it may be adapted for use for exploring other plant-pathogen interactions.
This protocol is designed for the assessment of disease on Arabidopsis thaliana leaves by the fungal pathogen Fusarium graminearum. The nuances of this protocol that have been troubleshot have encouraged us to publish this as its own entity. The protocol described has been adapted from previous protocols published by Machado-Wood et al. (2021) and Chen et al. (2003), as well as by taking several components from Cuzick et al. (2008). Loosely, this protocol details seed sterilisation and plant growth, inoculations and diseases assessment.
Image Attribution
All images included in this protocol were taken by Victoria Armer, Rothamsted Research.
Materials
Protocol specific materials
- Arabidopsis thaliana seed of desired genotype (NASC, UK)
- Murashige and Skoog (MS) Basal Salt media (Sigma Aldrich, UK)
- Deoxynivalenol (DON) (Sigma Aldrich, UK) * *please read and follow the COSHH data sheet because DON is toxic to life. (https://www.sigmaaldrich.com/GB/en/sds/sigma/d0156).
- Thin bleach (4.5% (v/v) Sodium hypochlorite)
- Tergitol (Sigma Aldrich, UK)
- 1M KOH
General lab consumables
1. 2ml microcentrifuge tubes
2. Toothpicks
3. dH2O
4. Agar
5. Square petri dishes (120mm x 120mm)
General plant growth consumables
1. Levington’s F2+S soil (Everris Ltd, UK)
2. Pots P7 (approx. volume 350cm3)
3. Plant trays
4. Capillary matting
5. Humidity lids
Equipment
1. Scales, weigh boats and spatulas
2. Stir bar
3. Duran flasks
4. Autoclave
5. pH meter
6. Fridge or cold room (5°C)
7. Flow hood
8. Controlled growth environment
8. Light meter
Safety warnings
Deoxynivalenol (DON) is a trichothecene mycotoxin that targets eukaryotic ribosomes, inhibiting protein translation. At chronic, low concentrations it can cause Leukaemia, and at higher, acute doses it induces nausea and vomiting, and can be fatal if swallowed. DON cannot be effectively destroyed by autoclaving. Please carefully read and follow the below COSHH data sheet and use appropriate PPE when handling DON.
1.1. Preparation of ½ MS plates for At germination
1.1. Preparation of ½ MS plates for At germination
This step was found necessary to eliminate plate contamination from other fungal and bacterial species during seed germination on ½ MS agar plates. Unlike usual ½ MS preparations, this one
does not contain sucrose to limit contamination.
This can be done ahead of time and stored at room temperature for a matter of weeks after autoclaving.
Prepare ½ MS media using the following recipe (makes 1000ml – scale as required):
a. 1.1 g MS
b. Up to 1000 mL with dH2O
c. Add stir bar and stir until suspended
d. Adjust to 5.7 with 1 Molarity (M) KOH
Aliquot into Duran flasks for autoclaving, removing stir bar
Add agar to1 % (v/v) in each flask (e.g. 2.5g for 250ml) and autoclave before use
Pour ½ MS media into square plates to a depth of 25mm for seed germination.
1.2. Sterilisation and plating of At seed
1.2. Sterilisation and plating of At seed
This was found to be the most ‘fool proof’ method of thorough seed surface sterilisation whilst ensuring high germination rates. N.B. don’t rush the serial dilution or miss steps – 5 washes ensures that the sterilisation solution is sufficiently diluted to not inhibit seed germination. Seed stratification in a cold room or fridge ensures even germination of At seed.
Create the sterilisation solution by mixing 50:50 dH2O and thin bleach (4.5% (v/v) sodium hypochlorite). This must be made fresh on day of use.
Aliquot required seed into 2ml microcentrifuge tubes and add 500 µL of the sterilisation solution. Invert to mix, ensuring all seed are suspended before settling and leave for 10 mins, inverting several times to ensure sufficient coating of seeds.
Conduct a dilution series of the bleach solution, pipetting out 450 µL of the sterilisation solution and replacing with 1000 µL of dH2O. Invert to suspend all seed and remove solution once seed settles. Repeat 4 times with subsequent additions of 1000 µL of dH2O to ensure sufficient dilution of the sterilising solution.
Pour seed onto an absorbent surface (e.g. blue roll) for pricking out onto ½ MS plates, at least 20mm apart, using (autoclaved) toothpicks.
Place ½ MS plates into a plastic bag and place in the dark in a cold room or fridge for 48hrs.
1.3. Plant Growth
1.3. Plant Growth
Plant growth
Plant growth can occur at neutral (12 hour) or long (16 hour) day length. We found that long day length worked well and sped up the experiment progression, allowing detached leaf inoculations to take place 4 weeks from germination.
Place square plates into a controlled growth room or cabinet with the following settings:
a. 16hr day length
b. 22 °C day temperature, 18 °C night temperature
c. 70% Humidity
d. 140µmol m-2 s-1 light intensity.
Leave to germinate for 7 days.
Seedling transplantation
Prepare trays and pots for seed transplantation by lining trays with capillary matting and filling P7 pots with Levington’s F2+S soil. Press until soil is firm and hydrate with dH2O until moist to touch on the surface. Leave space (i.e. cut out one pot from the tray) for easy watering from beneath to avoid disturbing delicate seedlings.
At 7 days, transplant At seedlings from ½ MS plates into F2+S using sterilised tweezers. Ensure roots are carefully tucked into the soil, pressing around the base of the developing rosette.
Spray the surface of the soil with dH2O and place a vent closed humidity lid over the tray, leaving under normal growth conditions for a further 14 days (Fig. 1).
Fig. 1. Growth conditions of Arabidopsis thaliana in trays of P7 pots. Image of 21-day old seedlings. (a) Example growth tray lined with capillary matting and (b) with humidity lid (vent closed) for plant growth.
At 21 days old (14 days after transplantation) remove the humidity lid and leave for another 7 days. Ensure soil is well hydrated throughout plant growth, watering from the bottom of the tray. Capillary matting should be moist to the touch and ensures even watering across the tray.
1.4. Detached leaf inoculations
1.4. Detached leaf inoculations
Detached leaf assay plates can be prepared a couple of days before inoculation if stored in a sealed bag in a cold room or fridge. The addition of Tergitol (a detergent and therefore fungal membrane stressor) encourages fungal growth on the At leaves rather than on the 1% water agar plates.
Preparation of 1% (w/v) water agar plates for detached leaf assay:
a. Add 4 g Agar to a 400ml Duran bottle
b. Top up to 400 mL with dH2O
c. Autoclave
d. Store at RT until use (can be prepared months in advance).
Melt 1% (w/v) water agar in the microwave (800W, 5.5 minutes)
Leave to cool until lukewarm
Add 200 µL Tergitol to each 400 mL 1% (w/v) bottle of water agar solution
Gently swirl flasks to disperse Tergitol without creating bubbles (about 20s of gentle motion)
Pour 1% (w/v) water agar plates to 25mm depth in flow hood and ensure plates are dry before use.
1.5. Rosette examples and leaf selection
1.5. Rosette examples and leaf selection
At 4 weeks old, At plants should have well-developed vegetative growth but have minimal evidence of apical bud development (Fig. 2).
Fig. 2. Example rosettes of At wild-type Col-0 and susceptible control min7 at 4 weeks under growth conditions described; scale bar = 20mm.
For the detached leaf assay, select leaves that are relatively small and flat for ease of inoculating, imaging and scoring. Leaves must be in active vegetative growth and not close to senescence – for easy identification, do not choose leaves that are particularly rounded indicating they originated in the early growth stages of the plant.
Cut leaves with sterile scissors and stick the petiole into the 1% (w/v) agar plate at a 45° angle. Ideally, leaves should not sit flush on the agar. Square plates can fit 6 leaves, spaced 30mm apart (Fig. 3).
Leaves should be selected and inoculated in quick succession i.e., leaves should be selected, cut and inoculated for each plate before moving onto the next plate.
Each plant is a biological replicate. Unless including technical replicates, only one leaf per plant is taken. The addition of Tergitol to the water agar prevents hyphal growth between replicates. The experiment should be repeated 3 times before conducting statistical analysis.
1.6. Inoculations
1.6. Inoculations
The addition of DON is a requirement to make infection of At with Fg occur routinely. Efforts to achieve a reliable and reproducible assay without the application of DON have been unsuccessful.
Selected leaves are gently scored on the agar plates using the end of a sterile 20µl (small) tip, either side of the midrib vein. This needs to be done very carefully to break through the adaxial epidermis layer, but not the palisade mesophyll layer. Several gentle strokes of the pipette tip may be required. This artificial wounding is necessary to allow F. graminearum to cause disease (Fig. 3.)
Fig. 3. Example square plate, filled with 1% water agar amended with Tergitol, laid with Arabidopsis WT Col-0 detached leaves. Wounding must damage the adaxial epidermis so that fungal infection can access the palisade mesophyll, but not penetrate the leaf entirely.
With gloved hands, drop 2µl of a 5x105 spores/ml water conidial suspension amended with 20 micromolar (µM) DON onto each wound. Appropriate controls should be used for each treatment to assess for the impact of scoring (dH2O) and DON only application (20µM DON).
Plates should be placed in the dark for 2 days in otherwise normal growth conditions and subsequently left for a further 5 days under low light (70µmol m-2 s-1 light).
N.B. this can be achieved by moving plates further from light sources and under cover. Use a light meter to ensure that light levels are appropriate. Low light levels prevent senescence of leaves, which interferes with disease assessment.
Detached leaves should be assessed at 7 days post infection (dpi), although particularly strong phenotypes may be visible at 4dpi in susceptible genotypes.
Fig.4. Example images of detached leaf assay at 7dpi for WT Col-0 and susceptible control min7.
Safety information
Deoxynivalenol (DON) is a trichothecene mycotoxin that targets eukaryotic ribosomes, inhibiting protein translation. At chronic, low concentrations it can cause Leukaemia, and at higher, acute doses it induces nausea and vomiting, and can be fatal if swallowed. DON cannot be effectively destroyed by autoclaving. Please carefully read and follow the below COSHH data sheet and use appropriate PPE when handling DON.
Follow COSHH data sheet for the safe disposal of unused DON.
1.7. Disease assessment
1.7. Disease assessment
Image analysis
Photographs of diseased images should be taken with a high-quality camera for recording purposes using a camera mount on a dark or light background.
Visual scoring
A DS index for visually scoring leaf disease severity is detailed by Chen et al. (2006) and offers a simple, reproducible way of assessing a large number of samples without downstream analysis.
Fungal burden
Fungal burden, the ratio of fungal to host gDNA, can be used to analyse the internal disease burden. A protocol for this is detailed in Machado-Wood et al. (2021), but briefly entails gDNA extraction followed by qPCR, with species-specific primers for quantification of entire gDNA.
DON mycotoxin analysis
The production of the DON mycotoxin by Fusarium graminearum can be measured as described in Cuzick, Urban and Hammond-Kosack (2008) using a commercially available enzyme-linked immunosorbent assay (ELISA). Ensure that the kit purchased matches the designated Fg chemotype of the strain to be used in the experiment; the WT reference strain PH-1 is a 15-ADON producer, an ester of deoxynivalenol (Miller, Taylor and Greenhalgh, 1983).
Protocol references
1. Alonso, J.M. et al. (2003). Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science. 301(5633):653-7. doi: 10.1126/science.1086391
2. Chen, X., Steed, A., Harden, C. and Nicholson, P. (2006). Characterization of Arabidopsis thaliana-Fusarium graminearum interactions and identification of variation in resistance among ecotypes. Mol. Plant Pathol. 7(5):391-403. doi: 10.1111/j.1364-3703.2006.00349.x
3. Cuzick, A., Urban, M., and Hammond-Kosack, K. (2008). Fusarium graminearum gene deletion mutants map1 and tri5 reveal similarities and differences in the pathogenicity requirements to cause disease on Arabidopsis and wheat floral tissue. New Phytol. 177(4): 990-1000. doi: 10.1111/j.1469-8137.2007.02333.x
4. Machado-Wood, A. K., Panwar, V., Grimwade-Mann, M., Ashfield, T., Hammond-Kosack, K. E. and Kanyuka, K. (2021). The vesicular trafficking system component MIN7 is required for minimizing Fusarium graminearum infection. J. Exp. Bot. 72(13):5010-5023. doi: 10.1093/jxb/erab170
5. Miller, J. D., Taylor, A., Greenhalgh, R. (1983). Production of deoxynivalenol and related compounds in liquid culture by Fusarium graminearum. Can. J. Microbiol. 29(9): 1171-1178. doi: 10.1139/m83-179