Key result
Virus-delivered short RNA inserts (24-, 28-, and 32-nt) successfully targeted conserved regions of functionally redundant gene pairs, triggering quantitative gene silencing in plant models.
Why the study?
Current VIGS protocols rely on 200-400 nt inserts in less conserved regions, but Nicotiana benthamiana allopolyploidy and functional redundancy motivated designing virus-delivered short RNA inserts to simultaneously target homeologous gene pairs.
Virus-delivered short RNA inserts (vsRNAi) as short as 24 nucleotides can effectively produce robust gene silencing phenotypes in model plants and crops, simplifying viral vector engineering for functional genomics.
Short vsRNAi constructs enable plant gene silencing; leaves open validation across crops and field conditions.
Engineered RNA viruses can downregulate target genes through virus-induced gene silencing (VIGS). Largely optimized using the model plant Nicotiana benthamiana , current VIGS protocols rely on viral vectors engineered to deliver 200-400 nt inserts with homology to a target gene, often located in less conserved regions to ensure specificity. Given the N. benthamiana allopolyploidy and functional redundancy, we leveraged enhanced genomics and transcriptomics resources to obtain curated locus annotations, which guided the design of virus-delivered short RNA inserts (vsRNAi) for simultaneous targeting of homeologous gene pairs. Our results showed that quantitative silencing phenotypes could be obtained using 24-, 28-, and 32-nt vsRNAi that targeted conserved regions of functionally redundant gene pairs. vsRNAi triggered informative transcriptome-wide changes, and target transcript downregulation was linked to gene-pair-specific production of 21- and 22-nt sRNAs. The portability of vsRNAi to crops was confirmed in tomato ( Solanum lycopersicum ) and scarlet eggplant ( Solanum aethiopicum ). Overall, our results support the usefulness of vsRNAi-based approaches in functional genomics and trait reprogramming of the model species N. benthamiana, as well as of crops that contribute to global and local food provisions. HIGHLIGHTS Comparative genomics enables design of virus-delivered short RNA inserts (vsRNAi) 32-, 28-, and 24-nt vsRNAi resulted in quantitative gene silencing phenotypes vsRNAi trigger transcriptome-wide changes for gene functional characterization vsRNAi elicit production of 21- and 22-nt small RNAs and gene downregulation vsRNAi approaches assist functional genomics and phenotypic alteration in crops
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García et al. (2025) studied this question. virus-delivered short RNA inserts (vsRNAi) was evaluated on quantitative gene silencing phenotypes and transcriptome-wide changes. Virus-delivered short RNA inserts (24-, 28-, and 32-nt) successfully targeted conserved regions of functionally redundant gene pairs, triggering quantitative gene silencing in plant models.
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