Materials and Reagents

Conical tubes (50 ml) (Corning, Falcon^®, catalog number: 352070 )
Parafilm M laboratory film (Hach, catalog number: 251764 )
Plastic Petri dishes with a transparent lid (JET BIOFIL cell and tissue culture plates 6 well)
Tips
Miracloth (EMD Millipore, catalog number: 475855 )
Labeling tape (Shamrock, catalog number: ST-12-20 )
Paper towels
Arabidopsis accessions: Columbia (Col-0)
Ethanol > 99.8% (Sigma-Aldrich, catalog number: 02860 )
Note: This product has been discontinued.
Lactic acid 85% (w:w) (Sigma-Aldrich, catalog number: L1250 , DL-Lactic acid 11.3 M)
Phenol (TE buffer equilibrated, pH 7.5-8.0) (Roti^®-Phenol, Carl Roth, catalog number: 0038.1 )
Glycerol ≥ 99% (Sigma-Aldrich, catalog number: G7757 )
Sterile distilled water (EMD Millipore, milli-q a10 water purification system)
Trypan blue (Sigma-Aldrich, catalog number: T6146 )
Potato dextrose agar (PDA) (BD, Difco^TM, catalog number: 213400 )
Potato dextrose broth (PDB) (BD, Difco^TM, catalog number: 254920 )
Sodium hypochlorite solution (Sigma-Aldrich, catalog number: 239305 )
Sodium dodecyl sulfate (SDS) 98% (Sigma-Aldrich, catalog number: 862010 )
Note: This product has been discontinued.
Tween 20 detergent (Sigma-Aldrich, catalog number: P1379 )
Trypan blue staining solution (see Recipes)
Potato dextrose agar (PDA) (see Recipes)
Bortytis cinerea (B.c) inoculum (see Recipe)
Potato dextrose broth (PDB) (see Recipes)
Seed sterilization solution (see Recipes)

Equipment

Micro scissors NOYE (Auxilab, catalog number: 62101111 )
Dissecting forceps, Toothless 125 mm (Auxilab, catalog number: 61302012 )
Growth chamber (Vötsch Industrietechnik, model: HERAEUS VB1514 )
Biological safety cabinet (Germfree, model: BBF-2SSCH )
Micropipets P1000, P200 and P20 from Rainin Instruments
Weight analysis 440 (KERN & SOHN)
Stereomicroscope (Leica Microsystems, model: MZ9 ) coupled to CCD camera (Leica Microsystems, model: DC 280 )
Conductimeter (HACHLANGE SPAIN, CRISON, model: BASIC30 )
Autoclave (JP SELECTA, model: MED 20 , catalog number: 4001757)

Software

ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2016)
Statgraphics Centurion XVI.II software (StatPoint Technologies, Inc. www.statgraphics.com)

Procedure

Growing Arabidopsis plants
1. Sterilize Arabidopsis seeds with the seed sterilization solution (see Recipes) for 20 min, then wash with sterile water four times.
2. Stratify seeds in 4 °C chamber for 2 days in darkness.
3. Germinate seeds in a mix of 3:1 (soil:vermiculite) in the growth chamber, for 21 days under 10 h light/14 h darkness photoperiod, with a light intensity of 120 lux at 22 °C and 60% humidity.
Inoculation with Botrytis cinerea
1. Sterilize all the material in an autoclave at 120 °C for 20 min.
2. Put the plants into a biological safety cabinet chamber and inoculate them with one drop of 5 μl of B.c fungal inoculum (see Recipes, Figure 1A) per leave.
3. Cover the plants with Parafilm (Figure 1B) to avoid dispersion of conidiospores and to maintain high humidity (90-100%) for optimal growth of the fungus. Return them to the growth chamber and make small holes in the Parafilm (> 10) with a yellow tip to avoid ethylene accumulation within the tray, which would activate plant defense response and stop fungal growth.
4. Incubate the B.c-inoculated plants in a growth chamber at 25 °C under a 10 h photoperiod until the day of sampling, usually 2 to 7 days post-inoculation (Figure 1C).
5. At each time-point of the experiment, harvest inoculated leaves with scissors and put them directly into plates containing 5 ml of fresh trypan blue solution (see Recipes, Figure 1D). At this point be sure that the leaves are completely immersed in the liquid; use a blue tip to submerge them if necessary. Cover the plate and wait for at least 30 min. A blue color will then be visible in the locations of dead tissue. Total staining time should not exceed 1 h. Avoid the use of boiling methods, vacuum infiltration methods or microwave treatments which will impede the later de-staining process.
  Note: Timing of sampling might change depending on Arabidopsis/accession/mutant/line-B.c interaction and fungal virulence.
6. Remove the trypan solution and re-fill the plates immediately with 98-100% ethanol. Seal the plates with Parafilm in order to avoid drying that might break the leaves or fix them to the plastic plate. Leave tissue in the ethanol solution at least overnight, then using a 5 ml pipet; replace the ethanol solution with fresh ethanol, repeatedly, until green tissue has become completely colorless (Figure 1E). Remove the ethanol and cover the leaves with 60% glycerol solution. At this point, the leaves are ready to be transferred to a glass surface to be visualized with a microscope (Figure 1F).
7. If necessary, cell death measurements might be validated by ion leakage losses using a conductometer according to Govrin and Levine (2000)* or measuring leaf death area with ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/ij/, 1997-2016).
  *Note: To measure ion leakage we followed the protocol of Govrin and Levine (2000). We used 5 inoculated or control leaves per biological replicate, and four replicates per genotype. Leaves were immersed into water for 3 h at room temperature and later conductivity was measured following manufacturer’s instructions. Total (100%) ion leakage was obtained from same samples maintained o/n at -80 °C. Data analyses were performed as is indicated below.
  
  Figure 1. Procedure of Arabidopsis leaf inoculation with B.c. A and B. Leaf surfaces of Arabidopsis lines were inoculated with 5 µl of fungus (10⁵-10⁸ spores/ml), and plants were transferred to a plastic tray with transparent lid to allow fungal growth. C. The progress of infection was followed until plant symptoms were observed. D. Leaves were harvested on plates and imbibed with trypan blue staining solution for no more than 1 h. E. Leaves were immersed in ethanol overnight until they were translucent. F. Leaves were transferred to 60% glycerol for microscopy purposes.
  
  Figure 2. Arabidopsis leaves with different degrees of infection. A-F. Photos taken immediately after cutting leaves from plants grown on soil. G-L. Photos of representative leaves stained with trypan blue, exhibiting levels of infection similar to the leaves shown above. Bar = 0.5 cm.

Data analysis

For all experiments at least ten leaves per ecotype are sampled to be stained. Experiments are repeated at least four times. When necessary, the stained leaf surfaces were measured with the software ImageJ (Particle Analysis, Surface Plot, and Measure Tools, following the user guide of ImageJ, https://imagej.nih.gov/ij/docs/pdfs/examples.pdf). Leaf ion leakage data were analyzed with the Statgraphics Centurion XVI.II software (StatPoint Technologies, Inc. www.statgraphics.com). Unifactorial analyses of variance (ANOVA) tests were performed, calculating the mean and standard error as well as Duncan’s comparison of means for a confidence level of 0.05%.

Recipes

Trypan blue staining solution
10 ml lactic acid (85% w:w)
10 ml phenol (TE buffer equilibrated, pH 7.5-8.0)
10 ml glycerol (≥ 99%)
10 ml of distilled water
40 mg of trypan blue (final concentration of 10 mg/ml)
Note: Store the solution at room temperature into a ventilation hood and always use fresh solutions.
Potato dextrose agar (PDA)
4 g/L PDA in distilled water
Botrytis cinerea (B.c) inoculum*
The Botrytis strain was grown at 28 °C on PDA medium plates (potato dextrose agar, Difco, Detroit, MI , USA) for eight days under darkness, and spores were collected in sterile water and stored in 20% glycerol at -80 °C until use.
*Note: The stock of the fungi should be preserved at -80 °C in 20% glycerol in order to avoid virulence loss caused by repeated growing of the fungus on plates. The final concentration of the fungus will depend on the virulence of the fungal isolate usually between 105-107 spores/ml. Fungal concentration (conidiospores per ml) is measured using a hemocytometer (http://www.hemocytometer.org/). When the B.c isolate has low virulence, or when high levels or rapid fungal growth are required, the inoculation may be performed in presence of potato dextrose broth medium (PDB) using the maximum dilution of PDB possible, adding the same concentration of PDB to control inoculated leaves.
Potato dextrose broth (PDB)
4 g/L PDB in distilled water
Seed sterilization solution
Hypochlorite solution (30%, v:v)
Sodium dodecyl sulfate (0.1%, v:v)

Acknowledgments

This protocol was modified from Keogh and collaborators (1980). We acknowledge M. Wilkinson from CGBP for his contribution. The research leading to these results received funding from No. AL13-P(I+D)-05, No. AL14- PID-26 to Domínguez J.A, and Berrocal-Lobo M, from RTA2013-00027-00-00 to Castellano M. M, and Berrocal-Lobo M.

References

Cooksey, C. J. (2014). Quirks of dye nomenclature. 3. Trypan blue. Biotech Histochem 89(8): 564-567.
Dean, R., Van Kan, J. A., Pretorius, Z. A., Hammond-Kosack, K. E., Di Pietro, A., Spanu, P. D., Rudd, J. J., Dickman, M., Kahmann, R., Ellis, J. and Foster, G. D. (2012). The Top 10 fungal pathogens in molecular plant pathology. Mol Plant Pathol 13(4): 414-430.
Gelder, M., Mayou, R. and Geddes, J. (2005). Psychiatry (3rd). Oxford pp: 238.
Govrin, E. M, and Levine, A. (2000). The hypersensitive response facilitates plant infection by the necrotrophic pathogen Botrytis cinerea. Curr Biol 10(13): 751-757.
Keogh, R.C., Deverall, B.J. and Mcleod, S. (1980). Comparison of histological and physiological responses to Phakopsora pachyrhizi in resistant and susceptible soybean. Transactions of the British Mycological Society 74(2):329-333
Lehmann, S., Serrano, M., L'Haridon, F., Tjamos, S. E. and Metraux, J. P. (2015). Reactive oxygen species and plant resistance to fungal pathogens. Phytochemistry 112: 54-62.
Lu, J. and Greco, M. A. (2006). Sleep circuitry and the hypnotic mechanism of GABA drugs. J Clin Sleep Med 2(2): S19-26.
Rolke, Y., Liu, S., Quidde, T., Williamson, B., Schouten, A., Weltring, K. M., Siewers, V., Tenberge, K. B., Tudzynski, B. and Tudzynski, P. (2004). Functional analysis of H(2)O(2)-generating systems in Botrytis cinerea: the major Cu-Zn-superoxide dismutase (BCSOD1) contributes to virulence on French bean, whereas a glucose oxidase (BCGOD1) is dispensable. Mol Plant Pathol 5(1): 17-27.
Salazar M, Peralta C, Pastor F. J. (2009). Tratado de Psicofarmacología. 2 Ed. Panamericana.
Salmon, A. G., Kizer, K. W., Zeise, L., Jackson, R. J. and Smith, M. T. (1995). Potential carcinogenicity of chloral hydrate--a review. J Toxicol Clin Toxicol 33(2): 115-121.
Silverman, J, and Muir, W. W., 3rd (1993). A review of laboratory animal anesthesia with chloral hydrate and chloralose. Lab Anim Sci 43(3): 210-216.