Environmental RNA interference (RNAi), initially discovered
in the nematode Caenorhabditis elegans, refers to the systemic gene
silencing in an organism induced by the uptake of environmental
RNAs [2]. Similar to C. elegans, the aggressive fungal pathogen,
Botrytis cinerea, is also able to take up RNAs from the environment
[3]. Further, a series of recent discoveries revealed a new mechanism of communication between plants and fungi. This phenomenon is known as cross-kingdom RNAi, in which plants send sRNAs
into the fungus to silence and target virulence genes, and in turn,
the fungus sends sRNAs into the plant host to target and silence
defense genes [4, 5]. Combined, these discoveries inspired the
development of spray-induced gene silencing (SIGS), a crop protection approach involving the foliar application of RNAs which
target and silence fungal pathogen genes.
Foliar application of RNAs has been a successful approach in
protecting several plants from fungal diseases, including barley
leaves from Fusarium graminearum [6], postharvest material
such as fruits and flowers from B. cinerea [3], and Brassica napus
plants from Sclerotina sclerotiorum [7]. The ability of these fungi to
take up environmental RNAs is crucial for RNA-based control
strategies to be effective. In order to establish best protocols for
using RNA to control specific fungal diseases, the uptake efficiency
of the fungi across types and treatments of environmental RNA
must be established. Additionally, research into fungal RNA uptake
may help to elucidate the mechanisms of cross-kingdom RNAi.
Here, we present a protocol for observing the uptake efficiency of
both double-stranded RNAs (dsRNAs) and small RNAs (sRNAs)
using B. cinerea as an example, though these methods can be easily
adapted across fungal species.
2 Materials
2.1 In Vitro Synthesis
of Fluorescein-Labeled
Double-Stranded RNAs
1. Phusion
® High-Fidelity DNA Polymerase.
2. 5Â Phusion HF buffer.
3. 2.5 mM dNTPs.
4. 10 μM Forward primer, with T7 promoter sequence
[5
0 -TAATACGACTCACTATAG-3
0 ] appended to 5
0 end.
5. 10 μM Reverse primer, with T7 promoter sequence
[5
0 -TAATACGACTCACTATAG-3
0 ] appended to 5
0 end.
6. Nuclease-free water.
7. Fluorescein RNA Labeling Mix Kit.
8. T7 RNA polymerase.
9. T7 RNA polymerase reaction buffer, 10Â, supplied with T7
RNA polymerase.
216
Rachael Hamby et al.
in the nematode Caenorhabditis elegans, refers to the systemic gene
silencing in an organism induced by the uptake of environmental
RNAs [2]. Similar to C. elegans, the aggressive fungal pathogen,
Botrytis cinerea, is also able to take up RNAs from the environment
[3]. Further, a series of recent discoveries revealed a new mechanism of communication between plants and fungi. This phenomenon is known as cross-kingdom RNAi, in which plants send sRNAs
into the fungus to silence and target virulence genes, and in turn,
the fungus sends sRNAs into the plant host to target and silence
defense genes [4, 5]. Combined, these discoveries inspired the
development of spray-induced gene silencing (SIGS), a crop protection approach involving the foliar application of RNAs which
target and silence fungal pathogen genes.
Foliar application of RNAs has been a successful approach in
protecting several plants from fungal diseases, including barley
leaves from Fusarium graminearum [6], postharvest material
such as fruits and flowers from B. cinerea [3], and Brassica napus
plants from Sclerotina sclerotiorum [7]. The ability of these fungi to
take up environmental RNAs is crucial for RNA-based control
strategies to be effective. In order to establish best protocols for
using RNA to control specific fungal diseases, the uptake efficiency
of the fungi across types and treatments of environmental RNA
must be established. Additionally, research into fungal RNA uptake
may help to elucidate the mechanisms of cross-kingdom RNAi.
Here, we present a protocol for observing the uptake efficiency of
both double-stranded RNAs (dsRNAs) and small RNAs (sRNAs)
using B. cinerea as an example, though these methods can be easily
adapted across fungal species.
2 Materials
2.1 In Vitro Synthesis
of Fluorescein-Labeled
Double-Stranded RNAs
1. Phusion
® High-Fidelity DNA Polymerase.
2. 5Â Phusion HF buffer.
3. 2.5 mM dNTPs.
4. 10 μM Forward primer, with T7 promoter sequence
[5
0 -TAATACGACTCACTATAG-3
0 ] appended to 5
0 end.
5. 10 μM Reverse primer, with T7 promoter sequence
[5
0 -TAATACGACTCACTATAG-3
0 ] appended to 5
0 end.
6. Nuclease-free water.
7. Fluorescein RNA Labeling Mix Kit.
8. T7 RNA polymerase.
9. T7 RNA polymerase reaction buffer, 10Â, supplied with T7
RNA polymerase.
216
Rachael Hamby et al.