Chapter 13
Labeling of dsRNA for Fungal Uptake Detection Analysis
Matteo Galli, Jafargholi Imani, and Karl-Heinz Kogel
Abstract
Double-stranded RNA (dsRNA) plays an essential role in many biological processes and has a great
potential for agronomic applications in disease and pest control. A simple and effective method to monitor
dsRNA uptake by fungi is crucial for the use of dsRNA as alternative fungicide. The protocol reported in
this chapter describes an efficient method to detect and localize labeled dsRNA in fungal hyphae. We use the
fungal Verticillium longisporum, a fungal plant pathogen that commonly infects rapeseed and other Brassica
species, to explain the procedure, though we have validated the method in a broad spectrum of fungi.
Hereafter we elucidate step-by-step the production, fluorescence labeling, as well as detection of dsRNA via
fluorescence microscopy in fungal mycelium.
Key words dsRNA detection, Fluorescent dsRNA, Environmental RNAi, Hyphae
1 Introduction
Double-stranded RNA (dsRNA) is an essential molecule in various
cellular processes, from eukaryotic organisms to viruses. In plants,
dsRNA is fundamental to triggering of gene silencing through
RNA interference (RNAi) through production of small RNAs
(sRNAs), including microRNAs (miRNAs) and small interfering
RNAs (siRNAs) [1, 2]. In this process, the dsRNA is cut into short
21–24 nucleotide (nt) RNA duplexes by DICER-like (DCL) endonuclease III enzymes. Subsequently, short RNA duplexes are
loaded onto ARGONAUTE (AGO) enzymes and associated proteins that together form the RNA-induced silencing complex
(RISC). One strand of the RNA duplex is discarded, while the
remaining guide strand guides the RISC to complementary
mRNAs through base-pairing. Upon matching, AGO cleaves the
target mRNA, causing its rapid degradation. Recent reports suggest that dsRNAs are also fundamental components controlling the
outcome of plant host-pathogen interactions. This phenomenon is
called cross-kingdom RNAi, where dsRNA is bidirectionally transferred between host and microbe to eventually target
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_13, © Springer Science+Business Media, LLC, part of Springer Nature 2020
227
Labeling of dsRNA for Fungal Uptake Detection Analysis
Matteo Galli, Jafargholi Imani, and Karl-Heinz Kogel
Abstract
Double-stranded RNA (dsRNA) plays an essential role in many biological processes and has a great
potential for agronomic applications in disease and pest control. A simple and effective method to monitor
dsRNA uptake by fungi is crucial for the use of dsRNA as alternative fungicide. The protocol reported in
this chapter describes an efficient method to detect and localize labeled dsRNA in fungal hyphae. We use the
fungal Verticillium longisporum, a fungal plant pathogen that commonly infects rapeseed and other Brassica
species, to explain the procedure, though we have validated the method in a broad spectrum of fungi.
Hereafter we elucidate step-by-step the production, fluorescence labeling, as well as detection of dsRNA via
fluorescence microscopy in fungal mycelium.
Key words dsRNA detection, Fluorescent dsRNA, Environmental RNAi, Hyphae
1 Introduction
Double-stranded RNA (dsRNA) is an essential molecule in various
cellular processes, from eukaryotic organisms to viruses. In plants,
dsRNA is fundamental to triggering of gene silencing through
RNA interference (RNAi) through production of small RNAs
(sRNAs), including microRNAs (miRNAs) and small interfering
RNAs (siRNAs) [1, 2]. In this process, the dsRNA is cut into short
21–24 nucleotide (nt) RNA duplexes by DICER-like (DCL) endonuclease III enzymes. Subsequently, short RNA duplexes are
loaded onto ARGONAUTE (AGO) enzymes and associated proteins that together form the RNA-induced silencing complex
(RISC). One strand of the RNA duplex is discarded, while the
remaining guide strand guides the RISC to complementary
mRNAs through base-pairing. Upon matching, AGO cleaves the
target mRNA, causing its rapid degradation. Recent reports suggest that dsRNAs are also fundamental components controlling the
outcome of plant host-pathogen interactions. This phenomenon is
called cross-kingdom RNAi, where dsRNA is bidirectionally transferred between host and microbe to eventually target
Manfred Heinlein (ed.), RNA Tagging: Methods and Protocols, Methods in Molecular Biology, vol. 2166,
https://doi.org/10.1007/978-1-0716-0712-1_13, © Springer Science+Business Media, LLC, part of Springer Nature 2020
227