**4. Successes of RNAi through nontransformative approaches**

The delivery of dsRNA through nontransformative approaches is likely to hit the market in four categories: (i) direct control agents; (ii) resistance factor repressors; (iii) developmental disruptors; and (iv) growth enhancers [9–11, 49–52]. As a direct control agent, nontransgenic approaches were successfully managed to achieve long-lasting gene silencing [9, 10, 18, 19, 41].

In some experiments, full-sized citrus and grapevine trees were treated with dsRNA using foliar sprays, root drenching, or trunk injections. Two hemipteran insects, a xylem- and a phloem-feeding, and a coleopteran chewing insect took up the dsRNA after feeding on plants previously treated with dsRNA [11, 28]. Similarly, rice plants were able to take up dsRNAs when their roots were soaked in dsRNA solution showing resistance against piercing-sucking and stem-borer pest insects [53] and also mites [15]. Altogether, these experiments are clear demonstrations that drench/soak roots, trunk injections, and sprays on leaves are success strategies for delivery of dsRNA molecules without any modification on plants DNA.-

Plant diseases caused by virus have a tremendous impact in food production and quality, being responsible for loses in several crops, fruits, and vegetables worldwide. Coherent with an ancient role to protect genome from invasive viruses, the RNAi mechanism can be reprogrammed to work by destroying any virus RNA. Without viral RNA, no viral proteins are made, thus preventing virus replication and plant diseases. Some studies have already been conducted on topical application of dsRNA to control plant viruses [41, 54]. However, a major limitation in the practical application of dsRNA to control viruses is that RNAs face a hostile environment where it is rapidly degraded with not only low uptake into plants, but also the short virus protection window of few days postspray. There are some rumors that the initiated pipeline branded "BioDirect" by Monsanto controls pest insects and plant viruses with sprays of dsRNA, but details on this probably are not publically available. To address some of these limitations, a layered double hydroxide (LDH) clay nanosheet, called "BioClay," was developed [55] and combined with dsRNA molecules. The clay nanoparticles are positively charged and so bind and protect the negatively charged RNAs; delivery occurs when atmospheric carbon dioxide and moisture react with clay nanoparticles breaking down LDH, gradually releasing RNAs. Using this dsRNA-LDH complex was possible to achieve long-lasting gene silencing, protecting tobacco plants from a virus for 20 days with a single spray [55, 56], thus extending the period of 5–7 days using naked dsRNA. The complex dsRNA-LDH protected plants in both local lesions and systematically. Also using RNAi to control plant viruses, the mechanism of dsRNA uptake into the leaf was investigated. It was reported a rapid systemic spread of dsRNA when leaves of *Nicotiana tabacum* cv. Xanthi were mechanically inoculated with naked dsRNA homologous to tobacco mosaic virus (TMV) [57]. From these experiments, we can conclude that topical application of dsRNA targeting virus genes can induce a systemic RNAi toward virus resistance.

Direct spray of dsRNA was also used experimentally to control the Colorado potato beetle (CPB), *Leptinotarsa decemlineata,* under greenhouse conditions [10]. The naked dsRNA molecules in foliar application were sufficiently stable for at least 28days, enough to control the CPB.-The authors also investigated the RNA degradation under UV light, where they concluded that an exposition of 1–2 h is needed for dsRNAs to become inactive in feeding assays. The long biological activity (28 days) during greenhouse feeding experiments suggests that naked dsRNA is more stable in leaf surface than in a glass surface used for the UV stability studies.

The fungi kingdom consists of a large and diverse group of eukaryotes, and plant diseases caused by fungi exert particular and agronomic impact on global grain and food production. Generally, the proteins dicer, argonaute, and RdRP, which are some of the major components of RNAi pathways, are present in most fungi species [58]. Therefore, the RNAi pathways can be harnessed to control plant diseases [22]. Sprays of CYP3-dsRNAs, targeting simultaneously three fungal ergosterol biosynthesis genes (P450 lanosterol C-14α-demethylases—*CYP51A, CYP51B, CYP51C*), on barley leaves were used to control *Fusarium graminearum* infections in the local areas, where dsRNA was sprayed, but strikingly also in unsprayed distal leaf parts, showing that dsRNA was systematically translocated within the plant [18]. The example above was a proof-of-concept article showing that after spray an even long dsRNA molecule (791 nt) could be taken up by the plant and transferred as unprocessed dsRNA via plant vascular system to infection sites, where it was processed by the fungal RNAi machinery to carry out its antifungal activity. The same authors also demonstrated that green fluorescent protein (GFP) from jellyfish was silenced in a *Fusarium graminearum* strain expressing GFP, suggesting that sprays of dsRNA are not sequence selective and thus with the potential for targeting any gene across several plant pathogens. Similar study [19] showed that dsRNA and sRNAs targeting dicer-like protein genes DCL1 and DCL2 of *Botrytis cinerea* were externally applied on fruits, vegetables, flower petals, and *Arabidopsis* leaves, followed with *B. cinerea*  infection. The authors showed that *B. cinerea* was able to take up dsRNA and sRNAs from the environment, inhibiting gray mold disease.

The study with full-sized citrus trees (2.5 m tall) was performed with 2 g of dsRNA in 15 l of water applied by root drench and injections [46]. The dsRNA was detected in psyllids and leafhoppers 5–8days postingestion from plants and for at least for 57days in the citrus trees; this allows the development of an area-wide pest suppression approach. Similarly, Koch et al. [18] showed that the CYP3-dsRNA labeled with the green fluorescent dye (ATTO 488) was detected in the vascular tissue 24 h after spraying leaves. Also, the leaf sections demonstrated that the fluorescence was detected in the xylem, in the apoplast and symplast of phloem parenchyma cells, companion cells, mesophyll cells, as well as in trichomes and stomata. The labeled dsRNA was detected also inside fungal conidia and germ tubes as well as in the fungal mycelium. These experiments conducted by Koch et al. [18] using sprays on barley leaf surface are the first examples of active dsRNA uptake by plant cells.-

The uptake of RNAs from the environment, a phenomenon known as environmental RNAi [8], has not yet been observed in mammals. This phenomenon was observed in *C. elegans*, others nematodes and insects [10, 59]. However, until recently, it was not clear whether plants and fungi could take up RNAs from the environment. From what we know so far, it is worth noting that plants and fungi are indeed capable to take up dsRNAs and sRNAs applied externally [18, 19]. As described above, the locally applied dsRNA can inhibit pathogen growth also at distal unsprayed leaves, so these RNAs should be able to spread systematically across plant cells and tissues [18, 55, 60]. The nematode *C. elegans* is able to take up environmental dsRNAs that are longer than 50 bp, where the shorter dsRNAs cannot be taken up [59]. Generally, plant-feeding insects are able to take up dsRNAs that are longer than 50–60 pb, but not shorter dsRNAs or sRNAs [61, 62], while fungi and plants can take up both external sRNAs and long dsRNAs [18, 19]. The differences in the uptake of RNA species between plants/fungi and insects suggest that entry/uptake channels or pathways may differ among organisms. In the light of this, the uptake mechanisms that the externally applied RNAs may be translocated into plant pathogens and/or herbivorous insects could have at least two possible routes for entry. First, for insects, RNAs could be directly taken up during herbivory or through the cuticle to get into insect cells; similarly for fungi, RNAs could be taken up directly into fungal cells after spray. Second, the RNAs could be taken up by plant cells first and then move into insect/fungal cells indirectly (**Figure 1**).

**Figure 1.** Two possible pathways of silencing insect and fungal genes induced by sprays of dsRNAs and sRNAs. There are at least two possible routes for RNAs to get into insect/fungal cells. Pathway 1: Insects and fungi directly take up sprayed RNAs. The up taken dsRNAs may be sliced into sRNAs by fungal or insect DCL proteins. Pathway 2: Externally applied dsRNAs and sRNAs are taken up by plant cells and then transferred into insect or fungal cells. The long dsRNAs may be sliced into sRNAs by plant dicer like (DCL). In both possible pathways, fungi take up longer and shorter dsRNAs, while insects take up dsRNAs longer than 50–60 bp in length. For phloem-feeding insects such as stinkbugs and aphids, sprayed RNAs may prove difficult to get into insect cell directly (pathway 1), while for chewing insects such as grasshoppers and caterpillar, RNAs are taken up easily during herbivory.

One obstacle, if not the biggest, is the cost for the mass production of dsRNA. While the issues of environmental stability and delivery are being addressed with creative innovations such as BioClay, making mass amounts of RNA is still expansive. Indeed, cost-effective methods will allow real-world applications of exogenous dsRNA for RNAi-mediated crop protection. To our knowledge, currently, there are no commercial RNAi-based products that utilize dsRNA as a spray for crop protection. Since the discovery of dsRNA and its potential for crop protection, some companies and academic scientists are seeking to develop more cost-efficient methods for large production of dsRNA. Similarly, RNAi to control devastating pests such as the Colorado potato beetle has obviously attracted attention in private research and development. As mentioned before, Monsanto (currently Bayer) and Syngenta (current ChemChina) have allocated major investments toward SIGS technology. Already in mid-2015, Monsanto launched its technology BioDirect, and although the principle was the same as we had seen in academia, these products work differently because they are not expressed in the leaves, but applied exogenously to the plants. Syngenta scientists also are developing lines of biocontrol products based on RNAi (https://www.youtube.com/embed/BiVZbAy4NHw?ecver=1). For example, these dsRNA-based products when sprayed onto the potato plants (field trials) or soy plants targeting genes of Colorado potato beetle and stink bug, *Nezara viridula*, respectively, suppress efficiently plant defoliation. Additionally, these products indicated that beneficial species even closely relate species that are not harmed [63].

The *in vitro* transcription and the *in vivo* syntheses are basically the two nonchemical sources of pure dsRNA with potential for mass production. Both strategies are based on annealing of two single-stranded RNAs (ssRNAs) enzymatically synthetized. Therefore, the annealing of ssRNAs may be performed *in vitro* [18, 19, 41, 57] or *in vivo* using bacterial cells deficient of enzyme- RNase III that degrades dsRNAs [35, 64, 65]; both approaches have advantages and disadvantages (**Table 1**). For example, there are possible hybridizations of two complementary ssRNA molecules that often result in a low final production of the correct and fully dsRNA duplexes.- Moreover, the *in vivo* production may contain bacterial homologous DNA molecules that will- affect RNA quality and its applicability.-

In the last few years, we have experienced an ever-growing interest in the market for dsRNA that has pushed long-established companies and startups toward better production, costefficient, and stable delivery systems. In instance, the cost to produce 1g of dsRNA (100 up to 800pb) has dropped from \$12,500 USD in 2008 to \$100 USD in 2016, and to less than \$60 USD today (July 2018) (http://www.agrorna.com/sub\_05.html). The agroRNA [67] produces bulk amounts of dsRNA that could be used in agriculture; however, it is worth noting that naked dsRNA as sold by agroRNA needs to be formulated if the objective is a long-lasting


**Table 1.** Common strategies\* for mass production of dsRNA with pros and cons.-

crop protection; otherwise, the dsRNAs will last only a few days. For crop protection, dsRNA does not need to be as pure as for medical application; however, at least for gene silencing in insects, the efficacy of dsRNA increased using purified RNA.-

Considering the rapid half-life of dsRNA mainly regulated due to action of RNases and sunlight in the hostile environment, a biotechnology company RNAagri (former APSE) developed a technology "Apse RNA Containers" (ARCs) that allows the mass production of encapsulated ready-to-spray dsRNA with costs near \$1 USD per 1g [68]. In brief, this technology is based on plasmids engineered to produce naturally occurring proteins such as capsids that are cotransformed with another plasmid coding for the target dsRNA with a sequence called the "packing site." The proteins produced by bacteria self-assemble around RNAs, resulting in RNA protected and resistant to environmental hostile conditions. For long-lasting crop protection with exogenous applications, the dsRNAs should be protected with coating of nanoparticles, liposomes, or polymers, which will increase the efficacy by reducing dsRNA degradation [9].

Alternatively to pure dsRNA, the *Escherichia coli* [HT115(DE3)] strain can be used to produce large quantities of dsRNA. The crude extracts of bacterially expressed dsRNA can be sprayed on crops to protect against pest insects and plant pathogens [9, 10, 35]. Also, symbionts have shown to be a promising dsRNA delivery method [69]. These naturally occurring organisms such as virus/bacteria can be engineered to generate a symbiont-mediated RNAi system to continue produce dsRNA in the host. In perennial crops, there is a risk that the viral/bacterial genome could lose the dsRNA construct and revert to the wild type, while for annual crops, the area could be treated once and then deliver dsRNA during the entire production season.

The virus-induced gene silencing (VIGS) has also a great potential [70–72] to transiently silence target genes of insects or pathogens on host plants. Therefore, if an insect or pathogenspecific RNAi inducer sequence is introduced into an engineered plant virus, siRNAs specific for insect/pathogen targets will be produced upon plant infection [18, 73].
