Transgene-Free, Virus-Based Gene Silencing In Plants By Artificial MicroRNAs Derived From Minimal Precursors

Artificial microRNAs (amiRNAs) are 21-nucleotide (nt) small RNAs computationally designed to specifically target sequence complementary RNA(s) and induce the silencing of the corresponding gene(s). The application of amiRNAs as biotechnological tools for functional genomics and crop improvement is limited by the need of transgenically expressing the amiRNA constructs in plants, as well as by the use of 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 AtMIR390a precursors, some of which included sequences from Oryza sativa MIR390, and concluded that highly effective and accurately processed amiRNAs can be produced from a chimeric precursor of only 86 nt. This minimal precursor was further validated in A. thaliana plants expressing amiRNAs against endogenous genes whose silencing led to visible and quantifiable phenotypes. Remarkably, minimal but not full-length precursors are able to 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 minimal amiRNA precursors are preferable to classic pri-miRNAs for expressing amiRNAs from viral vectors to induce whole-plant gene silencing in a transgene-free manner.