Materials and Reagents

Round (90 mm diameter) or square (120 x 120 mm) Petri dishes
Autoclavable, filtration material [e.g., Miracloth (Merck Millipore, catalog number: 475855 )]
Calcofluor white fluorescent brightener (Sigma-Aldrich, catalog number: F-3543 )
Congo red (Sigma-Aldrich, catalog number: 860956 )
Distilled water
Potassium hydroxide (KOH) (Sigma-Aldrich, catalog number: P5958 )
Glycerol (Sigma-Aldrich, catalog number: G5516 )
Potatoes
Cloth strainer
Potato dextrose agar (PDA) (Scharlab, catalog number: 01-483-500 )
Yeast extract (Merck Millipore, catalog number: 1119261000 )
Peptone (Merck Millipore, catalog number: 1119311000 )
Glucose (VWR, Normapur^®, catalog number: 50-99-7 )
Agar (BD, Bacto^TM, catalog number: 214010 )
MgSO₄·7H₂O (Merck Millipore, catalog number: 1058865000 )
KH₂PO₄ (Merck Millipore, catalog number: 1048731000 )
KCl (Merck Millipore, catalog number: 1049330500 )
NaNO₃ (Merck Millipore, catalog number: 1065371000 )
Sucrose (Merck Millipore, catalog number: 1076875000 )
Agar (Thermo Fisher Scientific, Oxoid^TM, catalog number: LP0011 )
2-(N-morpholino) ethanesulfonic acid (MES), monohydrate (Sigma-Aldrich, catalog number: 69892 )
Sodium hydroxide (NaOH) (Sigma-Aldrich, catalog number: S8045 )
Potato dextrose broth (PDB) (see Recipes)
CFW and CR stock solutions (see Recipes)
1. CFW stock solutions (20 mg/ml)
2. CR stock solutions (50 mg/ml)
Fungal growth agar medium (see Recipes)
1. YPD agar medium
2. PDA agar medium
3. Puhalla’s minimal agar medium (MM)

Note: CFW and CR are hazardous and potentially carcinogenic so caution must be taken to avoid skin contact or inhalation of these compounds.

Equipment

Protection gloves and coat
Flasks and magnetic stirrer for preparation of solutions
Neubauer chamber for cell counting
Microwave
Autoclave
Fungal growth incubator
Water bath
Laminar flow hood
Heat plate and cooking pot to boil potatoes
pH meter

Note: No other special equipment is required.

Software

SPSS 15.0 for Windows^® (LEAD Technologies Inc.)

Procedure

A major requirement for this protocol is to experimentally establish the optimum concentration of CFW or CR to be used. This may be influenced by several parameters, including size of the inoculum, genetic background of the strains and composition of the growth medium. The wild type strain used in our laboratory was Fusarium oxysporum f.sp. lycopersici (strain 4287/FGSC 9935). Appropriate CFW and CR concentration should be sublethal for the wild-type or reference strain used in the experiment. We typically define this parameter by inoculating serial dilutions of conidia in the form of spots on plates containing CFW and CR, with concentrations in the range of 10-200 μg/ml of CFW and 5-600 μg/ml of CR.

Production of fresh microconidia (3-4 d)
Inoculate 50 µl of a -80 °C stock microconidial suspension (in 30% glycerol) into 50 ml of liquid potato dextrose broth (PDB) (see Recipe 1). Grow cultures for 3-4 d at 28 °C with shaking at 170 rpm (Di Pietro and Roncero, 1998). Filter microconidia through one layer of Miracloth, centrifuge and wash with sterile water. Count conidia using a neubauer chamber and adjust to a final concentration of 1 x 10⁷conidia/ml.
Preparation of stock solutions of CFW (20 mg/ml) and CR (50 mg/ml) (1 h) (see Recipe 2)
Preparation of growth medium (1-2 h)
Prepare growth agar medium and autoclave (20 min at 120 °C). Cool the medium down to 50-60 °C in a water bath before adding CR and CFW to avoid any potentially harming fumes. Then, add CFW or CR to reach the desired concentrations. This can be done in a laminar flow hood. For F. oxysporum, we suggest to start by testing 4-5 different dye concentrations, in the following range: 10 to 200 μg/ml for CFW and 10 to 600 µg/ml for CR. Additionally, prepare a control plate without the dyes. The plates can be stored overnight, at room temperature and in the dark, to be used the next day.
Preparation of a serial dilution of microconidia (1-2 h)
Prepare a 10-fold serial dilution of freshly-obtained microconidia starting with the 1 x 10⁷ conidia/ml stock solution generated in step 1 (1 x 10⁷, 1 x 10⁶, 1 x 10⁵ and 1 x 10⁴ conidial/ml). Inoculate 2 µl drops of this series on plates containing CFW or CR and a control plate (final concentrations: 2 x 10⁴, 2 x 10³, 2 x 10² and 2 x 10 conidia/ml). We typically use square (120 x 120 mm) Petri dishes, to allow sufficient space for growth of the fungal colonies.
Incubation of plates (2-3 d)
Incubate plates at 28 °C in the dark for 2-3 d.
Interpreting and monitoring results
1. Sensitivity to CFW and CR should be determined by comparing the extent of colony formation of parental and mutant strains in control and CFW or CR plates. Colony diameter may be measured for quantification of differences in colony growth of the strains tested. For this, inoculate each single strain (a 5 µl drop of a 1 x 10⁷ microconia/ml suspension) on the control and cell wall stress plates, with 5 replicates each. We use round (90 mm diameter) Petri dishes. Incubate the plates at 28 °C for 5 d before scanning and measuring colony diameter.
2. Data from three independent experiments, each with five replicates, can then be analyzed with a statistic software such as SPSS 15.0 for Windows^®. Kruskal-Wallis analysis of variance (ANOVA) and Mann-Whitney test are used to assess statistically relevant differences among strains at P ≤ 0.05.

Representative data

Figure 1 shows the sensitivity of several F. oxysporum MAPK pathway mutants to CFW and CR (Perez-Nadales and Di Pietro, 2011). In F. oxysporum, the Fmk1 MAPK pathway is essential for plant infection (Di Pietro et al., 2001). This experiment revealed that Msb2, a signaling mucin receptor functioning upstream of Fmk1 and also the Fmk1 MAPK are required for cell wall integrity in F. oxysporum under these conditions.

Figure 1. Cell wall stress assay for F. oxysporum. A. Plates containing YPD medium or YPD medium supplemented with CR or CFW were spot-inoculated with the indicated strains and amounts of microconidia, incubated for three days at 28 °C and photographed. B. Colony diameter of the indicated media was measured at day 3 post-inoculation and plotted relative to the wild-type strain (100%). Error bars represent standard deviations calculated from five independent plates. Values with the same letter are not significantly different according to Mann–Whitney test (P ≤ 0.05).

Notes

During handling, CFW and CR solutions should always be protected from light by wrapping the containers in aluminum foil. As a precaution to prevent possible inactivation of CFW and CR by light, the plates should be incubated in the dark.

Recipes

Potato dextrose broth (PDB)
1. Boil 200 g of pealed potatoes in 1 L of distilled water for 60 min
2. Filtrate through a cloth strainer
3. Add 20 g of glucose and add up to 1 L distilled water
4. Sterilize by autoclaving 20 min at 120 °C
CFW and CR stock solutions
1. CFW stock solutions (20 mg/ml)
  We use the following diluting solution (Ram and Klis, 2006):
  20 mg/ml CFW
  0.5% (w/v) KOH
  83% (v/v) glycerol
  Note: We recommend preparing 20-30 ml of this solution prior to the experiment and store it at room temperature. On the day of the experiment, CFW can be freshly added to this solution reach a final stock concentration of 20 mg/ml.
2. CR stock solutions (50 mg/ml)
  Note: CR is in the di-sodium form and is readily dissolved in water.
  Prepare CR in sterile double-distilled water at a stock concentration of 50 mg/ml
Note: It is important to ensure that CFW and CR are completely dissolved by checking that no dye powder is visible in the stock solution after gentle mixing (i.e., in a rotatory shaker).
Fungal growth agar medium
The composition of the growth medium for this cell wall stress test may vary depending on the purpose of the experiment. We have used in our assays complete media such as YPD or potato dextrose agar (PDA) and Puhalla’s minimal medium (Puhalla, 1985). All media are prepared in distilled water.
1. YPD agar medium
  3 g/L yeast extract
  10 g/L peptone
  20 g/L glucose
  15 g/L Bacto agar
2. PDA agar medium
  3.9% PDA, w/v
3. Puhalla’s minimal agar medium (MM)
  0.5 g/L MgSO₄·7H₂O
  1 g/L KH₂PO₄
  0.5 g/L KCl
  2 g/L NaNO₃
  30 g/L Sucrose
  Add 15 g/L of oxoid agar
Note: For this assay, the growth medium is buffered to pH 5.5-7.0 before autoclaving (20 min at 120 °C) (Ram and Klis, 2006). For this, we add 50 mM MES to the growth medium solution and adjust to pH 6.5 with NaOH (a 10 N NaOH solution can be prepared in advance and be added slowly to the MES-buffered medium, until the desired pH is reached). After this, we add the agar (except for PDA), top up the solution to the desired volume with distilled water and autoclave it.

Acknowledgments

This research was supported by the SIGNALPATH Marie Curie Research Training Network (MRTN-CT-2005-019277) and by grants BIO2008-04479-E, EUI2009-03942, and BIO2010-15505 from the Spanish Ministerio de Ciencia e Innovación.

References

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