RNA silencing is a revolutionary innate immunity mechanism in eukaryotes that has greatly expanded our knowledge of gene expression and regulation in plants. RNA interference (RNAi) is an important regulatory mechanism that has become an invaluable tool for plant research, especially in terms of understanding the effects of gene regulation in response to abiotic and biotic stress. RNAi has enabled researchers to gain insight into gene function, pest resistance, and physiological processes in plants. Although RNA is known to play critical roles in biology, the extensive capabilities and complexity of this nucleic acid remained elusive and not fully understood. The intrinsic nature of the relatively labile, often single-stranded RNA molecule, and limited availability of RNA-dependent enzymes had slowed characterization and progress. Traditional research focused on messenger transcript (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA), but the universality of the molecule to life remained underestimated 
. The occurrence of viruses with RNA genomes (gRNA), the dominant genome type of viruses in plants, provided an extraordinary platform for the study of RNA function and gene expression. In a relatively brief period of time, knowledge of the dynamic RNA molecule has dramatically increased, and our understanding of RNA capabilities and applications rapidly expanded.
2. RNA Interference
Remarkably, transformation of plants with the genus Polerovirus
coat protein antisense RNA of the Potato leafroll virus produced similarly high levels of reduced virus titre and disease resistance as the corresponding sense mRNA 
. The response was rapid and all transformed plants exhibited sequence-specific sustained high levels of immunity regardless of the virus inoculum concentration (Figure 1
). Vector transmission of the virus by the green peach aphid Myzus persicae
was reduced and disease symptoms in foliage and tubers were eliminated. This showed that RNA was capable of conferring resistance as a trigger molecule and was subsequently observed in other plant virus groups 
. Replicative intermediates of RNA viruses include double-stranded RNA (dsRNA) and dsRNA secondary structures that are produced to regulate gene expression and are relatively stable compared to ssRNA due to the widespread occurrence of resilient ssRNA ribonucleases 
. The disease resistance achieved with antisense RNA demonstrated an inherent ability of RNA to protect against pathogens.
Figure 1. RNA interference (RNAi) against virus infection. Sense and antisense RNA protection against the single-stranded positive-sense RNA Potato leafroll virus (PLRV). Although phloem limited, members of the genus Polerovirus are transmitted in a persistent, non-propagative manner and cause considerable disease-related losses worldwide. The icosahedral virus is approximately 25 nm in size (top left transmission electron photomicrograph) and transmitted in an aphid-specific manner by the green peach aphid, Myzus persicae, approximately 2.5 mm in length (top middle scanning electron photomicrograph). Disease symptoms include stunting and chlorosis of infected plants (top right) that reduce yield and quality. Virus titres in plants expressing coat protein messenger RNA (mRNA, red line) or antisense RNA (aRNA, blue line) reduced virus levels significantly as compared to untransformed controls (green line), determined by double antibody sandwich (DAS) enzyme-linked immunosorbent assay (ELISA).
Experiments to transiently or stably increase endogenous gene expression often unexpectedly produced a decrease in mRNA. For example, attempts to overexpress chalcone synthase (CHS) in pigmented petunia petals blocked anthocyanin biosynthesis 
. Developmental timing and expression of the CHS mRNA by the endogenous gene was not altered but the level of transcript was reduced by 50 fold. This posttranslational gene silencing (PTGS) highlighted a regulatory mechanism of gene expression involving RNA interference. Polygalacturonase involved in plant cell wall degradation and ripening was inhibited in transgenic tomato expressing antisense RNA 
. Similarities between viral defense and gene silencing mechanisms suggested a common innate immunity in plants, including the systemic signalling in gene silencing contributing to the sequence-specific RNA interference 
3. Characterization of RNA Interference
RNA interference (RNAi) involves a sequence-specific suppression of gene expression by transcriptional or translational repression. The results of the RNAi characterization demonstrated that feeding or injecting gene-specific dsRNA into Caenorhabditis elegans
resulted in the disappearance of the targeted message 
. Silencing effects were observed with only a few molecules of unc-22
dsRNA per cell supporting a role as a trigger molecule. The RNAi mechanism is a naturally occurring process in most eukaryotes, conferring an ability of dsRNA to induce a sequence-specific systemic silencing process 
Exogenous dsRNA initiates RNAi by activating the ribonuclease Dicer enzymes that bind and cleave dsRNA into 21–24-base-pair small interfering RNA (siRNA) fragments with 3′ overhangs of 2–3 nucleotides (Figure 2
). Dicer proteins have an RNA helicase domain, RNase III motifs, and nucleic-acid-binding PAZ domain 
. The siRNA is converted to ssRNA when the sense complimentary RNA strand is degraded by Argonaute (AGO) enzymes and the antisense guide strand is incorporated into the RNA-induced silencing complex (RISC). Members of AGO possess a PAZ domain and a PIWI domain, resembling RNaseH, that are required for cleavage activity 
. The RISC complex further uses this strand to bind and degrade additional copies of sense complimentary RNA. Systemic silencing occurs and the inherit specificity suggests that nucleic acid is the signal molecule in plants 
. Amplification of even weak silencing signals indicates that RNA-dependent RNA-polymerase (RDRP) recognition and replication elicits effective silencing.
Figure 2. Mechanism of RNA interference (RNAi). RNAi is initiated by the enzyme Dicer that cleaves double-stranded RNA (dsRNA) into short fragments of approximately 21- to 24-nucleotide short interfering RNA (siRNA). The siRNA is unwound into single-stranded RNA and the sense RNA (green) is further cleaved and degraded by the enzyme Argonaute (AGO). The antisense RNA (red) is recruited into the RNA-induced silencing complex (RISC) that binds to the target sense RNA through the specificity of the complementary antisense RNA.
The occurrence of double-stranded RNA during viral RNA replication and hairpin RNA secondary structures regulating gene expression, indicated that ssRNA viruses have an inherent protective mechanism from RNAi 
. Silencing suppressors were subsequently identified within RNA virus genomes that targeted different components of RISC, such as the DICER-LIKE (DCL) proteins, and inhibit innate RNA silencing 
. Similar to the systemic nature of RNAi, silencing suppressors were also capable of systemic silencing suppression 
. The application of RNA silencing suppressors, such as the Tomato bushy stunt tombusvirus p19 protein, are often required in preventing PTGS in plant studies expressing homologous or heterologous genes 
4. Applications of RNA Interference
Stably or transiently expressed genes and nucleic acids in genetically engineered plants is often utilized in the study of gene function or the heterologous production of commercially valuable products. The use of full-length infectious clones (FLICs) of RNA viruses has facilitated the amplification of targeted genes, providing a convenient vector platform that can circumvent RNAi for site-directed mutations and increase or reduce gene expression to characterize PTGS and produce valuable heterologous commercial products. Application of virus-induced gene silencing (VIGS) has successfully utilized several RNA virus vectors including Tobacco rattle virus (TRV), Potato virus Y (PVY), TMV, and PLRV 
. Different virus vectors confer specific advantages such as titre and tissue specificity. For example, field-grown plants are subjected to strict containment by regulatory agencies to limit unexpected transmission in the environment by vectors. Phloem-specific expression by the PLRV FLIC is not transmitted mechanically or by vector when the capsid readthrough protein is replaced by the heterologous nucleic acid, eliminating accidental movement of genetic materials (Figure 3
Figure 3. RNA virus replication and applications. (a) Genomic and subgenomic RNA for the replication and translational strategies of the Potato leafroll virus, including a silencing suppressor produced immediately following virus disassembly. Replication involves the production of antisense RNA and subsequent sense subgenomic RNAs (sgRNAs) and expression of proteins involves several translational strategies including leaky start and stop codons, proteolytic site-specific cleavage of genomic RNA (gRNA), and an internal ribosomal entry site (IRES) sequence. (b) A full-length infectious clone (FLIC) of the Potato leafroll virus RNA amplifies expression of heterologous sequences for virus-induced gene silencing (VIGS) or production of commercially valuable proteins as shown (magnification 0.25×) with green fluorescent protein (GFP).
Innate immunity is more complex than originally envisioned and the RNAi regulatory mechanism is independent of other recognition and signalling pathways. Identification of genes for gene receptors and avirulence proteins has advanced our understanding of cellular resistance to a wide range of pathogens, including Pseudomonas syringae
, Cladosporium fulvum
, and Verticillium
. Mechanisms for signal amplification and recognition by receptors of sessile plants has improved our understanding of an important component of innate immunity 
. Cross-protection and intracellular communication has expanded with the discovery of RNAi and its role in innate immunity and gene regulation through extracellular plant and fungal RNA 
. Together the different sources of innate immunity provide complementary strategies in controlling historically devastating crop losses and emerging new threats to food production.
Exogenously introduced dsRNA to target plant pests began with the introduction of dsRNAs through microinjections 
. Microinjections are a favoured laboratory technique because incredibly precise amounts of dsRNA can be introduced into the target organism, allowing for precise delivery 
. Although an adequate delivery method for lab- and smaller-scale applications, microinjection unfortunately is not suitable for field-level control of plant pests and pathogens 
. Another delivery method involves the soaking of an organism in a suspension that contains the target dsRNA or directly spraying it with a solution containing the dsRNA 
. This method may not be as exact as microinjection; however, it is often used because of its ease of use and overall convenience. Many other methods of RNA delivery have been examined and application choice is often influenced by several factors including efficacy and economics.
Investigators have created transgenic plants that express desired dsRNAs to cause RNAi-induced gene silencing in the target organism when it ingests plant material, referred to as Host-Induced Gene Silencing (HIGS) 
. One example of HIGS was transgenic Zea mays
(corn) called SmartStax Pro that was created to target corn rootworm (Diabrotica vwirgifera virgifera
) that was approved for commercial use by the U.S. Environmental Protection Agency, the U.S. Food and Drug Administration, and the U.S. Department of Agriculture 
. Commercial acceptancet of transgenic plants has been challenging due to general public concerns related to genetic engineering, especially the stable insertion of nucleic acid from other organisms 
. A similar pest control efficacy was achieved with exogenously applied dsRNA in plants, representing a friendlier environmental and regulatory strategy for protection and production improvements 
Microorganisms transformed to contain target dsRNA have also been evaluated as a method for exogenous application. One notable example showed that bacteria transformed to contain the target dsRNA could be fed to insects to induce RNAi 
. These genetically modified bacteria in some cases were even able to colonize the gut of the host and continue to deliver dsRNA directly to it through the gut. Another example of the ingestion of a transformed microorganism is the study of transformed Saccharomyces cerevisiae
(yeast) containing dsRNA targeting spotted wing fruit fly Drosophila suzukii 
. This type of yeast naturally occurs on the surface of rotting fruit that D. suzukii
consumes, and therefore was seen as a viable vector to induce oral ingestion of the dsRNA. They had success and found that locomotor activity, survivorship, and reproductive fitness were all negatively impacted by the complimentary dsRNA 
. Acceptance of products derived from transgenic platforms are subjected to elevated regulatory and consumer acceptance concerns.