Engineering VIGS Vectors by Modifying Movement Proteins of the 30K Family

Abstract

Virus-induced gene silencing (VIGS) represents a particularly relevant tool in agricultural species for studying gene functionality. This study presents a novel approach for utilizing viruses belonging to the 30K family of movement proteins (MPs) as VIGS vectors. The method described here employs smaller inserts (54 bp or less) than those commonly used (100–500 bp). The developed strategy involves modifying 30K family MPs to introduce heterologous sequences of the gene of interest into their coding sequence. This approach enabled the successful induction of gene silencing in Nicotiana tabacum and Nicotiana benthamiana. Three representative viruses of the MP 30K family, alfalfa mosaic virus (AMV), cucumber mosaic virus (CMV), and tobacco mosaic virus (TMV) were employed. The capacity to induce gene silencing of small inserts (18–54 bp) was investigated, enabling to establish a correlation between insert size and silencing efficacy. This allowed the system to be calibrated to achieve partial silencing levels. The relationship between viral encapsidation and the level of gene silencing was also investigated, revealing that a high efficiency of viral encapsidation results in a reduction in the level of gene silencing achieved. Considering these findings, it can be concluded that the approach carried out with AMV, CMV, and TMV could be applied to other members of the MP 30K family. The MP 30K family comprises 20 viral genera and over 500 viral species, which can infect all agronomically significant plant species. Consequently, the strategy presented in this work could be applied to a wide range of relevant hosts.