3. Do not stop the reaction by heating, as this would reduce the
yield of sRNAs.
4. Adding 3 volumes instead of 2.2 volumes of 100% ethanol
improves the efficiency of sRNA precipitation.
5. To verify the sRNA quality and quantity, we suggest using a
20% PAGE gel instead of a highly concentrated agarose gel,
because PAGE gels are much more effective at sRNA separation and detection.
6. It is best to prepare MEA slides in a sterile petri dish by placing
one side of the microscope slide on the edge of the petri dish
and the other side down to the bottom. This creates a slope
that allows the liquid MEA medium to evenly flow down the
microscope slide when pipetting the MEA medium along the
upper side.
7. In the fluorescent dsRNA synthesis step, 2 μL of fluorescein
mix was added to the reaction, which was then eluted into
30 μL of dsRNA. Therefore, we recommend preparing the
fluorescein control by adding 1 μL of fluorescein into 15 μL
of nuclease-free water, and then to apply any dilution steps used
on the fluorescein-RNA mixture also to this control.
8. We recommend using around 400 spores total on each MEA
microscope slide, which will lead to growth of separated individual mycelium after 12 h. If too many spores are used,
individual mycelia could be difficult to distinguish, and if too
few spores are used, the mycelium can be difficult to find under
the microscope.
9. For the whole procedure in Subheading 3.3, work in sterile
conditions in a sterile hood to avoid the growth of bacteria on
the MEA microslides.
10. To correct for artificial/autofluorescence of fungal mycelium,
adjust the confocal microscope settings using water-treated
control samples of mycelium and use the same settings for
the rest of the samples.
References
1. Fisher MC, Hawkins NJ, Sanglard D, Gurr SJ
(2018) Worldwide emergence of resistance to
antifungal drugs challenges human health and
food security. Science 360(6390):739–742
2. Whangbo JS, Hunter CP (2008) Environmental
RNA
interference.
Trends
Genet
24
(6):297–305
3. Wang M, Weiberg A, Lin FM, Thomma BP,
Huang HD, Jin H (2016) Bidirectional crosskingdom RNAi and fungal uptake of external
RNAs confer plant protection. Nat Plants
2:16151
4. Weiberg A, Wang M, Lin FM, Zhao H,
Zhang Z, Kaloshian I, Huang HD, Jin H
(2013) Fungal small RNAs suppress plant
immunity by hijacking host RNA interference
pathways. Science 342(6154):118–123
5. Cai Q, Qiao L, Wang M, He B, Lin FM,
Palmquist J, Huang SD, Jin H (2018) Plants
send small RNAs in extracellular vesicles to
224
Rachael Hamby et al.
yield of sRNAs.
4. Adding 3 volumes instead of 2.2 volumes of 100% ethanol
improves the efficiency of sRNA precipitation.
5. To verify the sRNA quality and quantity, we suggest using a
20% PAGE gel instead of a highly concentrated agarose gel,
because PAGE gels are much more effective at sRNA separation and detection.
6. It is best to prepare MEA slides in a sterile petri dish by placing
one side of the microscope slide on the edge of the petri dish
and the other side down to the bottom. This creates a slope
that allows the liquid MEA medium to evenly flow down the
microscope slide when pipetting the MEA medium along the
upper side.
7. In the fluorescent dsRNA synthesis step, 2 μL of fluorescein
mix was added to the reaction, which was then eluted into
30 μL of dsRNA. Therefore, we recommend preparing the
fluorescein control by adding 1 μL of fluorescein into 15 μL
of nuclease-free water, and then to apply any dilution steps used
on the fluorescein-RNA mixture also to this control.
8. We recommend using around 400 spores total on each MEA
microscope slide, which will lead to growth of separated individual mycelium after 12 h. If too many spores are used,
individual mycelia could be difficult to distinguish, and if too
few spores are used, the mycelium can be difficult to find under
the microscope.
9. For the whole procedure in Subheading 3.3, work in sterile
conditions in a sterile hood to avoid the growth of bacteria on
the MEA microslides.
10. To correct for artificial/autofluorescence of fungal mycelium,
adjust the confocal microscope settings using water-treated
control samples of mycelium and use the same settings for
the rest of the samples.
References
1. Fisher MC, Hawkins NJ, Sanglard D, Gurr SJ
(2018) Worldwide emergence of resistance to
antifungal drugs challenges human health and
food security. Science 360(6390):739–742
2. Whangbo JS, Hunter CP (2008) Environmental
RNA
interference.
Trends
Genet
24
(6):297–305
3. Wang M, Weiberg A, Lin FM, Thomma BP,
Huang HD, Jin H (2016) Bidirectional crosskingdom RNAi and fungal uptake of external
RNAs confer plant protection. Nat Plants
2:16151
4. Weiberg A, Wang M, Lin FM, Zhao H,
Zhang Z, Kaloshian I, Huang HD, Jin H
(2013) Fungal small RNAs suppress plant
immunity by hijacking host RNA interference
pathways. Science 342(6154):118–123
5. Cai Q, Qiao L, Wang M, He B, Lin FM,
Palmquist J, Huang SD, Jin H (2018) Plants
send small RNAs in extracellular vesicles to
224
Rachael Hamby et al.