Chapter 12
Synthesizing Fluorescently Labeled dsRNAs and sRNAs
to Visualize Fungal RNA Uptake
Rachael Hamby, Ming Wang, Lulu Qiao, and Hailing Jin
Abstract
Fungal pathogens are responsible for severe crop losses worldwide. Defending crops against fungal disease
is critical for global food security; however, most current disease management approaches rely on chemical
fungicides that can leave dangerous residues in the environment. RNA interference (RNAi) is an important
process through which RNA molecules target and silence complementary genes, regulating gene expression
during both transcription and translation. Recently, it has been discovered that some species of fungi can
efficiently take up RNAs originating from their host plant and the environment. If these RNAs are
complementary to fungal genes, this can lead to the targeting and silencing of fungal genes, termed
“cross-kingdom RNAi,” if the RNA originated from a plant host, or “environmental RNAi,” if the RNA
originated from the environment. These discoveries have inspired the development of spray-induced gene
silencing (SIGS), an innovative crop protection strategy involving the foliar application of RNAs which
target and silence fungal virulence genes for plant protection against fungal pathogens. The effectiveness of
SIGS is largely dependent on the ability of fungi to take up environmental RNAs. Here, we describe the
protocols used to label and visualize RNAs which are taken up by Botrytis cinerea. This protocol could easily
be adapted for use across various fungal species. Determining the efficiency of RNA uptake by a specific
fungal species is a critical first step to determining if SIGS approaches could be an effective control strategy
for that fungus.
Key words Fungicides, RNA uptake, RNA interference, Environmental RNAi, Cross-kingdom
RNAi, dsRNA, RNA labeling, Spray-induced gene silencing (SIGS), Crop protection
1 Introduction
Plants are constantly under attack by pathogens, pests, and parasites. Of the various classes of plant pathogens, fungi are among the
most devastating. Alarmingly, drug resistance has been reported in
each major class of fungicide used in agriculture [1]. Due to this
and other reasons, the development of novel crop protection strategies is critical for global food security. The utilization of existing
pathways within the host or pathogen can be an effective method
for designing new approaches to combat disease.
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_12, © Springer Science+Business Media, LLC, part of Springer Nature 2020
215
Synthesizing Fluorescently Labeled dsRNAs and sRNAs
to Visualize Fungal RNA Uptake
Rachael Hamby, Ming Wang, Lulu Qiao, and Hailing Jin
Abstract
Fungal pathogens are responsible for severe crop losses worldwide. Defending crops against fungal disease
is critical for global food security; however, most current disease management approaches rely on chemical
fungicides that can leave dangerous residues in the environment. RNA interference (RNAi) is an important
process through which RNA molecules target and silence complementary genes, regulating gene expression
during both transcription and translation. Recently, it has been discovered that some species of fungi can
efficiently take up RNAs originating from their host plant and the environment. If these RNAs are
complementary to fungal genes, this can lead to the targeting and silencing of fungal genes, termed
“cross-kingdom RNAi,” if the RNA originated from a plant host, or “environmental RNAi,” if the RNA
originated from the environment. These discoveries have inspired the development of spray-induced gene
silencing (SIGS), an innovative crop protection strategy involving the foliar application of RNAs which
target and silence fungal virulence genes for plant protection against fungal pathogens. The effectiveness of
SIGS is largely dependent on the ability of fungi to take up environmental RNAs. Here, we describe the
protocols used to label and visualize RNAs which are taken up by Botrytis cinerea. This protocol could easily
be adapted for use across various fungal species. Determining the efficiency of RNA uptake by a specific
fungal species is a critical first step to determining if SIGS approaches could be an effective control strategy
for that fungus.
Key words Fungicides, RNA uptake, RNA interference, Environmental RNAi, Cross-kingdom
RNAi, dsRNA, RNA labeling, Spray-induced gene silencing (SIGS), Crop protection
1 Introduction
Plants are constantly under attack by pathogens, pests, and parasites. Of the various classes of plant pathogens, fungi are among the
most devastating. Alarmingly, drug resistance has been reported in
each major class of fungicide used in agriculture [1]. Due to this
and other reasons, the development of novel crop protection strategies is critical for global food security. The utilization of existing
pathways within the host or pathogen can be an effective method
for designing new approaches to combat disease.
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_12, © Springer Science+Business Media, LLC, part of Springer Nature 2020
215