A Method to Produce vsiRNAs in Plants with Cross-Kingdom Gene Silencing Capacity
2022
Hernán de Jesús Villanueva-Alonzo | Ana Paulina Haro-Álvarez | Arturo A. Alvarado-Segura | Raúl Enrique Valle-Gough | Juan Gualberto Collí-Mull | Alberto Cal-Torres | Víctor Ermilo Arana-Argáez | Julio César Torres-Romero | Oscar Alberto Moreno-Valenzuela | Geovanny Nic-Can | Benjamín Abraham Ayil-Gutiérrez | Karla Y. Acosta-Viana
Plants have evolved defense mechanisms to suppress viral transcription and replication by transcriptional and post-transcriptional gene silencing mediated by virus-derived small interfering RNAs (vsiRNAs). Based on this response, virus-induced gene silencing (VIGS)-based technology has been developed to silence target genes on either host plants or insect pests. This mechanism could also be used for the silencing of genes of interest in the medical field. We used the VIGS vector pEuMV-YP:Krt18, which was obtained by inserting the <i>Mus musculus</i> (<i>M. musculus</i>) Krt18 sequence into pEuMV-YP:ΔAV1. The objective was to evaluate the capacity of pEuMV-YP:Krt18 to induce <i>Nicotiana benthamiana</i> (<i>N. benthamiana</i>) production of vsiRNAs of a specific sequence that belongs to neither the plant genome nor the wild virus genome, which were used to induce cross-kingdom gene silencing between plants and mammals. The percentage of vsiRNA for each viral gene was calculated from an sRNA library of <i>N. benthamiana</i> plants infected by pEuMV-YP: Krt18. When the vsiRNAs were characterized, it was found that they corresponded to all the genes of the pEuMV-YP:Krt18 vector. These vsiRNAs induced the silencing of the Krt18 gene in <i>M. musculus</i> macrophages, supporting the ability to use VIGS vectors in plants as biofactories for the production of sRNAs that induce gene silencing in mammals.
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