Regulation of Plant Genes with Exogenous RNAs

Abstract

Exogenous RNA application, also known as spray-induced gene silencing (SIGS), is a new approach in plant biotechnology that utilizes RNA interference (RNAi) to modify plant traits. This technique involves applying RNA solutions of double-stranded RNA (dsRNA), hairpin RNA (hpRNA), small interfering RNA (siRNA), or microRNA (miRNA) directly onto plant surfaces. This triggers RNAi-mediated silencing of specific genes within the plant or invading pathogens. While extensively studied for enhancing resistance to pathogens, the application of exogenous RNA to regulate plant endogenous genes remains less explored, creating a rich area for further research. This review summarizes and analyzes the studies reporting on the exogenously induced silencing of plant endogenes and transgenes using various RNA types. We also discuss the RNA production and delivery approaches, analyze the uptake and transport of exogenous RNAs, and the mechanism of action. The analysis revealed that SIGS/exoRNAi affects the expression of plant genes, which may contribute to crop improvement and plant gene functional studies.

Keywords: RNA interference; dsRNA; exogenous RNAs; external application; gene silencing; miRNA; plant gene regulation; siRNA.

Figure 1

Exogenous application of dsRNA, siRNA, or hpRNA for plant gene regulation.

Figure 2

The representation of the in vitro and in vivo dsRNA production strategies for plant exogenous treatments. The strategies include enzymatic dsRNA synthesis using DNA-dependent T7 RNA polymerase, chemical synthesis, and bacterial and yeast dsRNA production systems. pL4440, pET28, pRSETa, pET17b, and phage phi6—vector systems used for dsRNA production in bacteria (Escherichia coli, Corynebacterium glutamicum, Bacillus thuringienesis, and Pseudomonas syringae). P406TEF1 and pRS426—vector systems used for dsRNA production in yeast (Yarrowia lipolytica and Saccharomyces cerevisiae).

Figure 3

Proposed mechanisms of exogenous RNA interference (exoRNAi) induction and down-regulation of a plant gene target. (a) The siRNAs are incorporated into the RISC that guides specific mRNA degradation (PTGS) of homologous mRNAs. (b) The components of the siRNA/mRNA complex can be amplified into secondary siRNAs through the activity of RDR6, a process known as transitivity. (c) RNA-directed DNA methylation is a mechanism where short RNA molecules guide DNA cytosine methylation to specific sequences, leading to transcriptional gene silencing (TGS). (d) Extensive alterations in the plant miRNA transcriptome after exogenous dsRNA application. The dashed arrows depict the different steps of the RNAi induction process and the movement of dsRNA/siRNA at the local level. Red arrows show the systemic movement of the RNA silencing signal in the treated plant. DCL—RNase III enzyme DICER-LIKE; AGO—Argonaute; RISC, RNA-induced silencing complex; RDR—RNA-dependent RNA polymerase; Pol V—DNA-dependent RNA polymerase V.