**3. Transformative versus nontransformative RNAi**

As aforementioned for field applications, the transformative RNAi includes the plant-incorporated protectants (PIPs; i.e., transgenic plants/RNAi-based plant traits), whereas non-PIP dsRNA-containing end-use products (dsRNA-EPs) might be formulated as dsRNA active ingredient such as a raw material for insecticide, antifungal, or antiviral with variable delivery modes (see Section 4).

The RNAi mechanism works at mRNA level exploring a sequence-dependent mode of action, which makes it unique in potency and selectivity compared to any regular agrochemicals. Therefore, one advantage of RNAi either by transformative or by nontransformative approach is that it would allow farmers to target pests more specifically. The technology can be designed by using sequences of RNA that match very specific gene sequences in a target pest; hence, RNAi should leave other species unharmed. The careful selection of unique regions of pest genes results in effects highly targeted, avoiding unintended effects.-

The transformative RNAi strategy through transgenic plants known as host-induced gene silencing (HIGS) has proved to be successful in the protection of crops against their specific pest insects [26, 36–38], plant pathogens [18, 19], viruses [22, 39–41], and nematodes [42, 43], recently reviewed [44].

A proof-of-concept milestone paper of Baum et al. [36] demonstrated that a dsRNA construct in a genetically engineered plant could provoke larval mortality in western corn rootworm (WCR), *Diabrotica virgifera*. This research was fundamental to spread the idea on the potential of dsRNA as a new pest control agent through transgenic plants. In September 2016, the Canadian Food Inspection Agency (CFIA) had announced the approval of the RNAi-based corn event Monsanto MON87411, the "SmartStax PRO" (expressing Cry3Bb1, Cry34Ab1/- Cry35Ab1, and DvSnf7) [38], containing a *D. virgifera* dsSnf7 construct in combination with two *Bt* constructs, for commercialization and release. Also, the US EPA had confirmed in June 2017 the approval of this event for commercial planting.

The development of a transgenic plant expressing dsRNA as a strategy for plant protection is straightforward, but it is not practical to every pest organism and crop [9, 10, 12]. Although the delivery of dsRNA by transgenic expression is well established, it requires generations of crop plants, which may take years and delays for practical application, depending on the regulatory rules, plant transformability, genetic stability, and public acceptance of genetically modified (GM) crop species [45].

While RNAi-based crops are expensive to produce and have a high risk of resistance breakdown, topical application is underway as a nontransformative approach that might enable RNAi-based pest management. Therefore, triggering an RNAi pathway in a pest organism may also be possible through a spray-induced gene silencing (SIGS) approach, without changing the plant DNA.-The SIGS as a nontransgenic approach for pest control was already published [46] as a proof-of-concept work and recently reviewed [9, 11]. Because of using this approach to silence genes without introducing heritable changes into the genome, it may not be regulated as a GM product. A dsRNA spray can be used almost immediately as a regular pesticide without having to go through several years involved in the development of a GM or conventionally bread crop. Besides, in several countries due to the slow regulatory procedure to approve transgenic crops, nontransformative RNAi strategies with similar results such as some of those demonstrated above could be applied. Still, the main drawback of nontransformative RNAi strategy is that as a plant grows, new leaves have to be sprayed to guarantee protection, so this implies in possible higher costs to farmers, whereas transgenic plants will produce dsRNA continuously. However, the vascular system of plants naturally translocates RNAs [47]. Therefore, sprays on leaves, injection in trunks, or soil application of dsRNA can travel long distances through plant vessels; hence, this can be exploited for the development of pest control strategies [11, 28].

The idea to use sprayable dsRNA was followed by an underlying supposition that this type of molecule would have a short half-life for an effective crop protection agent [48], and the short half-life of dsRNA in soil and by UV light has been confirmed [10]. This apparent challenge posed by SIGS approach is that the effects on plants last only a few days because unprotected- RNAs break down soon. Farmers may not want to apply extensive sprays to keep plants protected; however, there are some positive issues because the sprays of dsRNA can be quickly tailored toward a pest organism, much faster than a GM crop, and last only a few days or weeks- different from most regular pesticides. Crop protectors should bear in mind that there is no- need for a pest control agent persist active for months to become an efficient pest control agent.-

Regardless of the target species, for a successful nontransformative RNAi strategy, it is of paramount importance to identify unique regions in very essential target genes, so that brief changes in the level of expression can provoke severe consequences as well as delivery of sufficient amount of intact dsRNA.-Alternatively to transgenic plants, the delivery of dsRNA can- be through other routes including dsRNA sprays, dsRNA expression in bacteria, trunk injection, and engineered viruses, among others. For example, to control plant viruses, farmers are obligated to either grow varieties with resistance to viruses or try to kill the organism that spread, such as aphids or hemipterans. Sprays with dsRNA might be rapidly tailored against existing or new type of virus, and the gene-silencing effects of RNAi will last only a few days,- enough to suppress virus replication. Overall, the SIGS approach opens up a range of possibilities for several pest insects that are difficult to control such as root-feeding and sap-feedinginsects, plant viruses, and plant pathogens, especially in perennial crops (e.g., fruits such as grapes, apples, and citrus), where plant transformation takes years to develop and is costly.
