corresponding genes in a sequence-specific manner. In this way,
dsRNAs positively or negatively regulate genes involved in plant
defense or fungal virulence, respectively [3–5]. Successful translational research has led to agronomic application of this natural
phenomenon. Crop protection techniques have been established,
where artificial dsRNA is either expressed in plants to target interacting pathogens or pest (host-induced gene silencing, HIGS) or
applied directly on plants by spraying (spray-induced gene silencing, SIGS), also called environmental RNAi [6, 7]. Both technologies have been already used to control a vast number of pathogens,
including Botrytis cinerea, Verticillium dahliae, Sclerotinia sclerotiorum, Fusarium spec., and pests, including Diabrotica virgifera
virgifera [8–12, 14]. Nonetheless, not all plant pathogens can be
targeted and controlled through HIGS or SIGS. A recent example
is Zymoseptoria tritici, a major fungal pathogen of wheat: all current
RNAi-mediated crop protection techniques failed to protect wheat
plants against this fungal pathogen [13]. Hence, for further analysis
of the amenability of a fungal species to disease control by dsRNA,
it is of vital importance to investigate its ability to take up and
process exogenous dsRNA. The method described in detail in this
section portrays one possible approach to easily produce, deliver,
and detect labeled RNA duplexes in germinating fungal mycelia.
We present here a case study on Verticillium longisporum (Vl), an
economically important fungal plant pathogen of Brassicaceae.
Application of the labeling strategy described herein indicates a
strong ability of this fungus to take up dsRNA.
2 Materials
Prepare all solutions and buffers using deionized water at room
temperature (RT). Prepare and store all reagents at RT (unless
indicated otherwise by the manufacturer). The waste of residual
materials should be in accordance with safety regulations.
2.1 Fungus Culture
and Propagation
1. Verticillium longisporum wild-type strain Vl41 (Institute of
Plant Pathology and Crop Protection, Georg August University, Go ¨ttingen, Germany).
2. Potato dextrose broth (PDB): 20 g/l Glucose, 4 g/l potato
extract, pH 5.6 Æ 0.2 at 25
C (see Note 1).
3. Potato dextrose agar (PDA): 20 g/l Glucose, 4 g/l potato
extract, 17 g/l agar, pH 5.6 Æ 0.2 at 25
C (see Note 1).
4. Sterilized lab ware (scalpel, bottles, flasks, cork borer, glass
funnel, and Petri dishes).
5. Orbital flask shaker (3 μmol photons/m
2 /s).
6. Fungal growing chamber (70 μmol photons/m
2 /s).
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Matteo Galli et al.
dsRNAs positively or negatively regulate genes involved in plant
defense or fungal virulence, respectively [3–5]. Successful translational research has led to agronomic application of this natural
phenomenon. Crop protection techniques have been established,
where artificial dsRNA is either expressed in plants to target interacting pathogens or pest (host-induced gene silencing, HIGS) or
applied directly on plants by spraying (spray-induced gene silencing, SIGS), also called environmental RNAi [6, 7]. Both technologies have been already used to control a vast number of pathogens,
including Botrytis cinerea, Verticillium dahliae, Sclerotinia sclerotiorum, Fusarium spec., and pests, including Diabrotica virgifera
virgifera [8–12, 14]. Nonetheless, not all plant pathogens can be
targeted and controlled through HIGS or SIGS. A recent example
is Zymoseptoria tritici, a major fungal pathogen of wheat: all current
RNAi-mediated crop protection techniques failed to protect wheat
plants against this fungal pathogen [13]. Hence, for further analysis
of the amenability of a fungal species to disease control by dsRNA,
it is of vital importance to investigate its ability to take up and
process exogenous dsRNA. The method described in detail in this
section portrays one possible approach to easily produce, deliver,
and detect labeled RNA duplexes in germinating fungal mycelia.
We present here a case study on Verticillium longisporum (Vl), an
economically important fungal plant pathogen of Brassicaceae.
Application of the labeling strategy described herein indicates a
strong ability of this fungus to take up dsRNA.
2 Materials
Prepare all solutions and buffers using deionized water at room
temperature (RT). Prepare and store all reagents at RT (unless
indicated otherwise by the manufacturer). The waste of residual
materials should be in accordance with safety regulations.
2.1 Fungus Culture
and Propagation
1. Verticillium longisporum wild-type strain Vl41 (Institute of
Plant Pathology and Crop Protection, Georg August University, Go ¨ttingen, Germany).
2. Potato dextrose broth (PDB): 20 g/l Glucose, 4 g/l potato
extract, pH 5.6 Æ 0.2 at 25
C (see Note 1).
3. Potato dextrose agar (PDA): 20 g/l Glucose, 4 g/l potato
extract, 17 g/l agar, pH 5.6 Æ 0.2 at 25
C (see Note 1).
4. Sterilized lab ware (scalpel, bottles, flasks, cork borer, glass
funnel, and Petri dishes).
5. Orbital flask shaker (3 μmol photons/m
2 /s).
6. Fungal growing chamber (70 μmol photons/m
2 /s).
228
Matteo Galli et al.