4. Forty-eight hours after inoculation of labeled dsRNA, transfer
10 μl of conidia suspension onto a glass slide and take images of
growing mycelia in darkness with a fluorescence microscope
using appropriate filtering forAtto488 imaging (excitation at
482–514 nm and emission at 520 nm). Take pictures with
either a fluorescent microscope equipped with a 10Â/20Â
objective (Plan-Apochromat 10Â/20Â 0.8) (see Fig. 3a, b),
or a confocal microscope equipped with the 488 nm laser line
of an argon multiline laser (11.5 mW) and a water immersion
objective (HCX APO L40x0.80 W U-V-l objective) (see
Fig. 3c–f).
4 Notes
1. For PDB and PDA production, mix the ingredients with 1 L
dH 2 O and autoclave for 15 min at 121
C with a pressure of
approximately 15 pounds per square inch (PSI). To make PDA
plates, pour the still hot (~50
C) PDA medium into Petri
dishes under sterile conditions. Petri dishes can be stacked
one on top of another to dissipate the condensation heat.
2. TRIzol is toxic to skin and can give nausea if inhaled. Use
TRIzol only with gloves and under a chemical hood.
3. Digital images can be processed with imaging processing software (e.g., Adobe Photoshop) to optimize brightness, contrast, and color and to enable an overlay of the
photomicrographs.
4. In our case we selected the Vl41 Cytochrome P450 monooxygenase (VlCYP1) as target for labeled dsRNA. The VlCYP1
mRNA sequence is not available in the literature. The closest
conceivable homologous sequence is the Verticillium dahliae
Cytochrome P450 monooxygenase (VdCYP1; VDAG_05890 in
VdLs.17 genome; 11). Therefore, after analyzing VdCYP1 for
efficient siRNA hits (see Fig. 1), we amplified with primers
based upon VdCYP1 sequence a 475 nt fragment of VlCYP1
(see Fig. 2), which supposedly had the highest number of
efficient siRNA hits. The primers (a) and the VlCYP1 partial
sequence used as the template to generate the labeled dsRNA
(b, 228 efficient siRNA hits) are listed below:
(a) VlCYP1 primers:
l
VlCYP1_F: 5
0 - AGAACCGCCTCATTGAGCAT-3
0
l
VlCYP1_R: 5
0 - ATCGCTCGGGATCAAATACCT-3
0
Labeling of dsRNA for Fungal Uptake Detection Analysis
235
10 μl of conidia suspension onto a glass slide and take images of
growing mycelia in darkness with a fluorescence microscope
using appropriate filtering forAtto488 imaging (excitation at
482–514 nm and emission at 520 nm). Take pictures with
either a fluorescent microscope equipped with a 10Â/20Â
objective (Plan-Apochromat 10Â/20Â 0.8) (see Fig. 3a, b),
or a confocal microscope equipped with the 488 nm laser line
of an argon multiline laser (11.5 mW) and a water immersion
objective (HCX APO L40x0.80 W U-V-l objective) (see
Fig. 3c–f).
4 Notes
1. For PDB and PDA production, mix the ingredients with 1 L
dH 2 O and autoclave for 15 min at 121
C with a pressure of
approximately 15 pounds per square inch (PSI). To make PDA
plates, pour the still hot (~50
C) PDA medium into Petri
dishes under sterile conditions. Petri dishes can be stacked
one on top of another to dissipate the condensation heat.
2. TRIzol is toxic to skin and can give nausea if inhaled. Use
TRIzol only with gloves and under a chemical hood.
3. Digital images can be processed with imaging processing software (e.g., Adobe Photoshop) to optimize brightness, contrast, and color and to enable an overlay of the
photomicrographs.
4. In our case we selected the Vl41 Cytochrome P450 monooxygenase (VlCYP1) as target for labeled dsRNA. The VlCYP1
mRNA sequence is not available in the literature. The closest
conceivable homologous sequence is the Verticillium dahliae
Cytochrome P450 monooxygenase (VdCYP1; VDAG_05890 in
VdLs.17 genome; 11). Therefore, after analyzing VdCYP1 for
efficient siRNA hits (see Fig. 1), we amplified with primers
based upon VdCYP1 sequence a 475 nt fragment of VlCYP1
(see Fig. 2), which supposedly had the highest number of
efficient siRNA hits. The primers (a) and the VlCYP1 partial
sequence used as the template to generate the labeled dsRNA
(b, 228 efficient siRNA hits) are listed below:
(a) VlCYP1 primers:
l
VlCYP1_F: 5
0 - AGAACCGCCTCATTGAGCAT-3
0
l
VlCYP1_R: 5
0 - ATCGCTCGGGATCAAATACCT-3
0
Labeling of dsRNA for Fungal Uptake Detection Analysis
235