# A Method to Produce vsiRNAs in Plants with Cross-Kingdom Gene Silencing Capacity
## 1 Introduction  
RNA interference (RNAi) is a sequence-specific gene silencing process mediated by small RNAs (sRNAs) of 18 to 24 nucleotides (nt). Depending on their biogenesis, small RNAs can be classified into small interfering RNAs (siRNAs), microRNAs (miRNAs), or virus-derived interfering RNAs (vsiRNAs) [1; 2; 3]. RNAi regulates endogenous and exogenous gene expression at the transcriptional and post-transcriptional levels [1; 4]. Briefly, the gene silencing mechanism consists of (i) the formation of double-stranded RNA (dsRNA), (ii) cutting of dsRNA into sRNA of 18-25 nt by the DICER enzyme family, (iii) methylation of sRNA (in plants), (iv) sRNA incorporation into the RNA-induced silencing complex (RISC), (v) recognition of target sequence, and (vi) mRNA degradation or inhibition of transcription or translation [5]. This mechanism occurs in most eukaryotic cells, as has been shown in pioneering studies in animals [6], plants [7], and fungi [8]. This process has an impact on cell differentiation and division and defense against the stress of biotic and abiotic origin [9; 10].  
The ability of sRNAs to mediate gene silencing is not limited to the cell that produces it but can be transported to other cells and even among organisms from different kingdoms. miRNA168a present in rice has been reported to be found in the blood serum of people who consume rice daily. On the other hand, it has been shown that when miRNA168a is included in the mouse diet, silencing of the gene that codes for the low-density lipoprotein receptor is induced [11]. Other plant miRNAs have therapeutic potential; for example, the honeysuckle plant (_Lonicera japonica_) is used in traditional Chinese medicine, whose active substance is miRNA2911, which can inhibit the replication of influenza A and SARS-CoV-2 viruses [12; 13]. Other therapeutic properties attributed to plant miRNAs are anticarcinogenic [14; 15; 16], anti-inflammatory, and immunomodulatory properties of the immune system [17].  
Therefore, the use of plant miRNAs is a promising alternative to combat diseases in mammals. However, these applications are limited to only some plant miRNAs, which naturally have a target with therapeutic potential; alternatively, transgenic plants can be used to generate artificial miRNA (amiRNA) with therapeutic use. Another possibility, which does not require the use of transgenic plants and is technically less complex, is based on the use of the plants' antiviral system, which is the approach addressed in the present investigation.  
The vsiRNAs are chemically similar to miRNAs. It has been shown that in some viral infections in plants, vsiRNAs are produced to trigger gene-silencing mechanisms against viral genomes [4; 18; 19; 20]. From this defense mechanism of plants, virus-induced gene silencing (VIGS) vectors have been designed to induce the silencing of host genes, substituting part of the viral genome for the sequence of the host gene that is intended to be silenced [21].  
Unlike the use of the VIGS vector previously described, we explored the possibility of inducing specific vsiRNAs in _Nicotiana benthamiana_ (_N. benthamiana_) for a mammalian gene and using them to silence the gene in _Mus musculus_ (_M. musculus_). For this, we used the VIGS vector pEuMV-YP:\(\Delta\)AV1 based on the _Euphoria Mosaic Virus_ (Euphoria Mosaic Virus Yucatan Peninsula; EuMV-YP) [22], to which, as a study model, we included a fragment of the Cytok-18 (Krt18) gene from _M. musculus_, which is expressed in macrophages and is not present in _N. benthamiana_. The resulting vsiRNA profile showed that these are distributed throughout the pEuMV-YP:Krt18 genome with greater accumulations for derivatives of the BC1, AC3, AC2, and AC1 genes. The production of Krt18 gene-derived vsiRNA was detected in new tissues, including leaves that develop after pruning the plant. We demonstrated the ability of VIGS using pEuMV-YP:Krt18 to yield vsiRNA, which enables the induction of Krt18 gene silencing in cultures of _M. musculus_ macrophages (cross-kingdom silencing).