Abstract
Virus-induced gene silencing (VIGS) allows for the rapid targeting of gene expression and has been instrumental in characterizing plant genes. However, foreign sequences inserted into VIGS vectors are rarely maintained for unknown reasons. Citrus yellow vein-associated umbravirus-like virus (CY1) with its solved secondary structure was converted into a VIGS vector to determine why simple hairpins inserted into non-functional, single-stranded locations are not maintained. When CY1 contained foreign hairpins with thermodynamic properties (positional entropy and/or ΔG) differing from those of natural CY1 hairpins, deletions arose within a few weeks of infecting Nicotiana benthamiana. In contrast, duplication and insertion of four natural CY1 hairpins (up to 200 nt) into the same locations were retained until plant senescence. Hairpins containing similar conformations and thermodynamic properties as natural hairpins were also retained, as were hairpins that shared thermodynamic properties but were conformationally distinct. By predicting and modulating these thermodynamic properties, a hairpin was retained by CY1 for at least 30 months in citrus. These findings strongly suggest that RNA viruses have evolved to contain substructures with specific thermodynamic properties, and hairpins containing these properties are stable when inserted into non-functional regions of the genome, opening up VIGS for long-lived trees and vines. IMPORTANCE: Plus-strand RNA plant viruses are used as tools to introduce small interfering RNAs (siRNAs) into laboratory plants to target and silence genes. However, virus-induced gene silencing (VIGS) vectors engineered to contain foreign hairpins or other sequences for siRNA generation are not stable, and the foreign sequences are rapidly lost. We found that foreign sequences are not maintained in an umbravirus-like VIGS vector (CY1) because their physical properties conflict with the innate properties of the CY1 genome's substructures (i.e., hairpins). When natural CY1 hairpins were duplicated and inserted into locations where previous inserts were rapidly lost, the hairpins were now stable as were unrelated hairpins with the same physical properties. By mimicking the physical properties of the viral genome, one insert was stable for over 30 months. These results suggest that RNA viral genomes have evolved to have specific physical properties, and these properties appear to be similar for other plus-strand RNA viruses.