# A Method to Produce vsiRNAs in Plants with Cross-Kingdom Gene Silencing Capacity
## 4 Discussion  
The VIGS vector remained infective in _N. benthamiana_ after adding the Krt18 fragment, observing that plant symptoms were similar to those previously reported [22]. It has been reported that the VIGS method can induce gene silencing in host and insect pests using vsiRNA. This is achieved by replacing part of the viral genome with the sequence of interest while preventing any loss of the infective capacity [22]. Gene silencing level is due to the nature of the host and the virus, the stem-like structures that form their mRNA, and the sequence length. For instance, in Begomoviruses, this needs to be between 90 and 800 bp for optimal results [27]. In this study, the vsiRNAs of interest were those derived from a 432 bp fragment of the _M. musculus_ Krt18 gene. The dsRNA generated during viral infection [19; 28] is subsequently processed by RNAse III, called Dicer (DCL 1-4), to generate siRNAs mainly from 18-24 nt in size. The siRNA derived from pEuMV-YP:Krt18 were mostly 21, 22, and 24 nt, which is consistent with reports for Begomovirus and other viral infections, in which DCL4, DCL2, and DCL3 activity produce 21, 22, and 24 nt siRNA, respectively [29; 30].  
Begomovirus-derived vsiRNAs can be distributed evenly between the two components (DNA-A and DNA-B) of the viral genome, although, on occasion, the accumulation of one component can be greater [19]. In the present results, the vsiRNA derived from pEuMV-YP:Krt18 accumulated equally between the two components. This is potentially useful in increasing the production of vsiRNA since the B component can be used for VIGS vector modification [27].  
Generally, vsiRNAs are derived from all viral genes [25]. In infections with a Begomovirus such as the African Cassava Mosaic Virus, the greatest vsiRNA accumulation corresponds to gene overlap regions [31]. We found that the vsiRNA distribution in the viral genome was similar to what was previously reported [25; 31]. Our results agree that the vsiRNAs had greater accumulation in the overlapping genes (Figure 2g), and the expression level of viral genes can influence the greater accumulation of derived vsiRNAs. Additionally, another factor that influenced the greater accumulation of vsiRNA (hotspots) was the % GC (Figure 2h), which coincides with what was reported [3].  
In order to estimate the concentration of vsiRNA generated, a region of the Krt18 sequence with a Tm of 60 \({}^{\circ}\)C was randomly chosen. The accumulation of vsiRNA derived from the Krt18 sequence at 28 dpi was 0.386 pmol/g from fresh tissue. This quantification corresponded to vsiRNA 5\({}^{\prime}\)-CCAGACCGAGAAGGAGGACCAUG-3\({}^{\prime}\). However, other vsiRNAs derived from the Krt18 sequence were also found (Figure 2g), so the total concentration of vsiRNA was higher. In addition, a diverse group of vsiRNA sequences can be produced for the same gene, which is more effective for gene silencing than a single sequence [32]. pEuMV-YP:Krt18-derived vsiRNAs efficiently induced Krt18 gene silencing in _Mus musculus_ macrophages. This suggests that plants infected with a VIGS vector could be used for the production of vsiRNA for some therapeutic purposes, similar to how some medicinal plants are used, whose active substance is miRNA [12; 13; 16]. VIGS vectors can be applied to edible plants so that decoctions or consumption of these plants through the diet can be used for therapeutic purposes since mammals can absorb plant miRNA through the gastrointestinal tract and, at the same time, maintain their ability to induce gene silencing in the different organs of the animal [16; 33]. However, in vivo studies are still needed. Because of the diversity and quantity of vsiRNAs that are produced by this strategy, it would be interesting to investigate if there is a silencing of additional genes. This could be performed by mRNA sequence analysis of vsiRNA-treated and untreated cells.