Transgene-free, virus-based gene silencing in plants by artificial microRNAs derived from minimal precursors

Artificial microRNAs (amiRNAs) are highly specific, 21-nucleotide (nt) small RNAs designed to silence target transcripts. In plants, their application as biotechnological tools for functional genomics or crop improvement is limited by the need of transgenically expressing long primary miRNA (pri-miRNA) precursors to produce the amiRNAs in vivo. Here, we analyzed the minimal structural and sequence requirements for producing effective amiRNAs from the widely used, 521-nt long AtMIR390a pri-miRNA from Arabidopsis thaliana. We functionally screened in Nicotiana benthamiana a large collection of constructs transiently expressing amiRNAs against endogenous genes and from artificially shortened MIR390-based precursors and concluded that highly effective and accurately processed amiRNAs can be produced from a chimeric precursor of only 89 nt. This minimal precursor was further validated in A. thaliana transgenic plants expressing amiRNAs against endogenous genes. Remarkably, minimal but not full-length precursors produce authentic amiRNAs and induce widespread gene silencing in N. benthamiana when expressed from an RNA virus, which can be applied into leaves by spraying infectious crude extracts. Our results reveal that the length of amiRNA precursors can be shortened without affecting silencing efficacy, and that viral vectors including minimal amiRNA precursors can be applied in a transgene-free manner to induce whole-plant gene silencing.