Development of a Ligation-Independent Cloning-Based Dual Vector System for RNA Interference in Plants.

RNA interference (RNAi) is an evolutionarily conserved post-transcriptional gene silencing mechanism that responds to double-stranded RNA (dsRNA) by sequence-specific downregulation of target genes. The dsRNA-mediated RNAi technology has become one of the most widely used and powerful tools for functional genomic studies in diverse organisms. However, its application has been limited due to the technical difficulty of making RNAi constructs caused by the inverted repeat structure that is required for the formation of hairpin RNA. Here, we present a ligation-independent cloning-based dual vector-mediated RNAi system for silencing specific genes in plants. This approach is simple, efficient, and cost-effective and can be readily adapted to other binary vectors for functional analysis of target genes and the development of sustainable disease and pest control strategies in a broad range of plant species.

Potato Virus X-Based microRNA Silencing (VbMS) In Potato.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has been developed and applied for various plant species including Nicotiana benthamiana, tomato, Arabidopsis, cotton, and monocot plants such as wheat and maize. Here, we describe a detailed protocol using PVX-based VbMS vectors to silence endogenous miRNAs in potato. To knock down the expression of a specific miRNA, target mimic (TM) molecules of miRNA of interest are designed, integrated into plant virus vectors, and expressed in potato by Agrobacterium infiltration to bind directly to the endogenous miRNA of interest and block its function.