RNA Interference Past and Future Applications in Plants

Antisense RNA was observed to elicit plant disease resistance and post-translational gene silencing (PTGS). The universal mechanism of RNA interference (RNAi) was shown to be induced by double-stranded RNA (dsRNA), an intermediate produced during virus replication. Plant viruses with a single-stranded positive-sense RNA genome have been instrumental in the discovery and characterization of systemic RNA silencing and suppression. An increasing number of applications for RNA silencing have emerged involving the exogenous application of dsRNA through spray-induced gene silencing (SIGS) that provides specificity and environmentally friendly options for crop protection and improvement.


Introduction
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 [1]. 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. This review summarizes key developments in the discovery and characterization of RNAi and examines applications in plants to advance agricultural biotechnology, crop engineering, pest control, and virus resistance.
Plant viruses can cause major diseases in crops worldwide. Management has relied on a combination of strategies involving virus eradication and transmission prevention. Early studies reported the occurrence of cross-protection in plants where infection with a mild strain of a virus protected against infection by more pathogenic strains [2]. A major advance based on this phenomenon, and the suggestion that introduction of the viral coat protein may provide protection, was the resistance observed with the introduction of the Tobacco mosaic virus (TMV) coat protein mRNA into transgenic tobacco [3]. Several subsequent studies reported a similar resistance for other groups of plant single-stranded RNA (ssRNA) viruses including the family Solemoviridae, one of the most devastating group of viruses worldwide for many important crops [4,5]. Molecular characterization group of viruses worldwide for many important crops [4,5]. Molecular characterization and control of Solemoviridae has been especially challenging as they are phloem-limited and transmitted in a persistent circulative manner by specific aphids.

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 [4,6]. 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 [7]. 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 [8]. The disease resistance achieved with antisense RNA demonstrated an inherent ability of RNA to protect against pathogens.  Experiments to transiently or stably increase endogenous gene expression often unexpectedly produced a decrease in mRNA. For example, attempts to overexpress chalcone 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 [9]. De-velopmental 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 [10]. 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 [11,12].

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 [13]. 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 [6,9,10,13].
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 [7]. 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 [7]. 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 [7,12]. Amplification of even weak silencing signals indicates that RNA-dependent RNA-polymerase (RDRP) recognition and replication elicits effective silencing. synthase (CHS) in pigmented petunia petals blocked anthocyanin biosynthesis [9]. 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 [10]. 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 [11,12].

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 [13]. 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 [6,9,10,13].
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 [7]. 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 [7]. 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 [7,12]. Amplification of even weak silencing signals indicates that RNA-dependent RNA-polymerase (RDRP) recognition and replication elicits effective silencing.  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 [14,15]. 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 [16]. Similar to the systemic nature of RNAi, silencing suppressors were also capable of systemic silencing suppression [17]. 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 [14].

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 [18][19][20][21]. 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).
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 species [22][23][24][25]. Mechanisms for signal amplification and recognition by receptors of sessile plants has improved our understanding of an important component of innate immunity [26,27]. 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 [28,29]. 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 [30,31]. Microinjections are a favoured laboratory technique because incredibly precise amounts of dsRNA can be introduced into the target organism, allowing for precise delivery [32]. Although an adequate delivery method for laband smaller-scale applications, microinjection unfortunately is not suitable for field-level control of plant pests and pathogens [30,31]. 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 [32]. 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 (Table 1).
several RNA virus vectors including Tobacco rattle virus (TRV), Potato virus Y (PVY), TMV, and PLRV [18][19][20][21]. 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).  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) [31,33]. 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 [31,33,34]. 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 [33,35]. 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 [30].
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 [36]. 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 [37]. 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 [37]. Acceptance of products derived from transgenic platforms are subjected to elevated regulatory and consumer acceptance concerns. Tomato, lettuce, rose, and grape DCTN1, SAC1, DCL1, and DCL2 Drop inoculation of dsRNA for lettuce, rose, tomato fruit, and grape, dsRNA spray for tomato leaves [64] Rapeseed Peroxidase activity, TIM44, thioredoxin reductase, pre-40s ribosomal particle, necrosis-inducing peptide 1 Detached leaves sprayed with dsRNA [65] Tomato and chickpea DCL1 and DCL2, VPS51, bik1, and SAC1 dsRNA spray [66] Hyaloperonospora arabidopsis Arabidopsis Hpa-CesA dsRNA and sRNA added to spore inoculation [67] Phakopsora pachyrhizi Soybean Acetyl-CoA acyltransferase 40S ribosomal protein S16, glycine cleavage system H protein Chinese cabbage leaf discs were soaked in guanidine coated polymer that coated dsRNA and were fed to larvae [106] Spodoptera litura Castor Bt toxin receptor dsRNA injected into early 5th-instar larvae [107] Spodoptera frugiperda Corn sfVATPase, sfKIF, sfCDC27 Larvae fed dsRNA suspension [108]

Spray-Induced Gene Silencing
Application of RNAi as a foliar spray has become of particular interest, specifically because it has promise in being turned into a viable and specific biopesticide that would be available for commercial use. This technology is referred to as Spray-Induced Gene Silencing (SIGS), and it is a method that allows non-transformative control of plant pests and pathogens [31,35]. This mechanism involves the application of long siRNAs and dsRNAs through a foliar spray to the affected host, which will induce RNAi when consumed by the target pest or pathogen. This mechanism can allow for specific control of harmful pathogens, without some of the downstream effects that a chemical-based pesticide may have on the surrounding ecosystem [35]. To date, SIGS has been successful in treating a wide range of pathogens and pests (Table 1). One major drawback of this technique is that ssRNAs and even dsRNAs are relatively unstable, especially when exposed to the elements [31,35]. Considerable effort surrounding the SIGS strategy is going into testing various coatings and methods to stabilize the RNAs to allow for higher efficiency [35].
Published exogenously applied RNA studies report efficacies of up to 100% with over 30 days of activity and were influenced by many parameters including the genetic target, RNA size, and environment (Table 1). With all of these successes, however, there are always places for improvement and many roadblocks that continue to pose challenges with SIGS. To begin with, dsRNA has relatively short environmental survival, as mentioned previously, but may be an advantage from a regulatory perspective as the molecule does not persist and contaminate the environment. Some ways of increasing this efficiency include the use of liposomes or nanoparticles as transfection agents, or even the chemical modification of one or both strands of the dsRNA [30]. Polymeric nanoparticles have been synthesized and used because of their overall stability, ease of surface modification, their biodegradability, and environmental safety. A popular polymer to use is one called chitosan, which is often used because of its relatively cheap cost, non-toxicity, and general biodegradability [30]. Taning et al. [121] reported that the use of a cationic liposome branded Lipofectamine to coat the dsRNA resulted in a 40-50% efficiency of gene-silencing. Without the use of the transfection agent, they were unsuccessful in their goal to induce gene silencing through RNAi. Chemical modifications are not often used due to their high cost and general safety concerns, but they can be used to improve molecule stability, increase double-stranded siRNA half-life in vivo, target siRNA to specific cells, and many other functions [30].

Conclusions
RNA silencing is an essential component of innate immunity and gene regulation in plants and a rapidly growing number of tools are available for applications. Efficacy and survivability of the dsRNA is being actively explored to meet specific environmental and industry requirements. Exogenous RNA survivability and uptake may be improved using polymeric, lipid-based, and inorganic nanoparticles (Table 1). Advances made in synthetic biology have opened new possibilities for optimizing traits by modulating metabolic and immunity pathways via siRNA delivery. Systemic cellular and plant movement of triggers and signals observed with the RISC response in a plant facilitates protection in tissues not exposed to the dsRNA [12,17]. As research continues on this fascinating technology, new possibilities for applications in agricultural improvements continue to emerge, such as creating improved crops that have higher yields and greater nutritional value, resistant to pests and disease, resilient to environment and climate changes, and higher yields and quality. Future developments are expected in the application of RNAi technology in plants and subsequently other biological organisms. For instance, research into controlling epigenetic elements in gene silencing will have important implications for both agricultural biotechnology and fundamental evolutionary studies [122]. There are still many exciting possibilities for future development and applications of RNA interference in the areas of crop improvement, pest management, and human health care therapies.

Conflicts of Interest:
The authors declare no conflict of interest.