2.3 Labeled dsRNA
Production and In Vitro
Assay Detection
1. Fungal conidia (Vl41).
2. Spore-counting chamber (e.g., Fuchs-Rosenthal, Germany).
3. Bright-field microscope.
4. Si-Fi v21 software (siRNA finder, http://labtools.ipkgatersleben.de).
5. Primers flanked by T7 promoter sequences (5
0 -TAATACG
ACTCACTATAGGG-3
0 ).
6. Atto 488 RNA Labeling Kit (Jena Bioscience, Germany).
7. Silica-based membrane columns for RNA purification.
8. RNA wash buffer: 1 mM EDTA, 10 mM Tris–HCl, ethanol
80% V/V, pH 8.
9. DNase/RNase-free water.
10. RNase-free 1.5 mL tube.
11. 96 Microtiter well plates.
12. Microscope glass slides.
13. Fluorescence microscope.
14. Digital image processing software (see Note 3).
3 Methods
In the following, the fungus V. longisporum Vl41 is taken as an
example to demonstrate the labeling strategy and to demonstrate
the ability of a fungus to take up dsRNA. Nevertheless, the technique described here can be applied to a vast array of fungi [7, 11,
13 , 14].
Carry out all procedures at RT unless otherwise specified.
3.1 Fungal Vl41
Culture and Selection
of the Target Template
1. Vl41 culture: Prepare a fungal stock on PDA Petri dish plates
and incubate at 25
C in 16/8-h light period (light intensity:
70 μmol photons/m
2 /s). For maintenance, the fungus should
be routinely subcultured within an interval of 3 weeks on new
Petri dishes or stored in the dark at 4
C. In sterile conditions,
punch an agar block (diameter 0.5 cm
2 ) with a sterilized cork
borer, transfer the agar block to a freshly prepared PDA plate,
and seal the plate with parafilm.
2. Vl41 broth culture: All steps should be performed under sterile
conditions. Punch an agar block (diameter 0.5 cm
2 ) with a
sterilized cork borer and transfer it into flasks containing PDB
medium. Incubate on an orbital shaker (100 rpm) under constant illumination from one near-white-light tube (3 μmol
photons/m
2 /s) at RT.
230
Matteo Galli et al.
Production and In Vitro
Assay Detection
1. Fungal conidia (Vl41).
2. Spore-counting chamber (e.g., Fuchs-Rosenthal, Germany).
3. Bright-field microscope.
4. Si-Fi v21 software (siRNA finder, http://labtools.ipkgatersleben.de).
5. Primers flanked by T7 promoter sequences (5
0 -TAATACG
ACTCACTATAGGG-3
0 ).
6. Atto 488 RNA Labeling Kit (Jena Bioscience, Germany).
7. Silica-based membrane columns for RNA purification.
8. RNA wash buffer: 1 mM EDTA, 10 mM Tris–HCl, ethanol
80% V/V, pH 8.
9. DNase/RNase-free water.
10. RNase-free 1.5 mL tube.
11. 96 Microtiter well plates.
12. Microscope glass slides.
13. Fluorescence microscope.
14. Digital image processing software (see Note 3).
3 Methods
In the following, the fungus V. longisporum Vl41 is taken as an
example to demonstrate the labeling strategy and to demonstrate
the ability of a fungus to take up dsRNA. Nevertheless, the technique described here can be applied to a vast array of fungi [7, 11,
13 , 14].
Carry out all procedures at RT unless otherwise specified.
3.1 Fungal Vl41
Culture and Selection
of the Target Template
1. Vl41 culture: Prepare a fungal stock on PDA Petri dish plates
and incubate at 25
C in 16/8-h light period (light intensity:
70 μmol photons/m
2 /s). For maintenance, the fungus should
be routinely subcultured within an interval of 3 weeks on new
Petri dishes or stored in the dark at 4
C. In sterile conditions,
punch an agar block (diameter 0.5 cm
2 ) with a sterilized cork
borer, transfer the agar block to a freshly prepared PDA plate,
and seal the plate with parafilm.
2. Vl41 broth culture: All steps should be performed under sterile
conditions. Punch an agar block (diameter 0.5 cm
2 ) with a
sterilized cork borer and transfer it into flasks containing PDB
medium. Incubate on an orbital shaker (100 rpm) under constant illumination from one near-white-light tube (3 μmol
photons/m
2 /s) at RT.
230
Matteo Galli et al.