RNAi as a Foliar Spray: Efficiency and Challenges to Field Applications

Abstract

RNA interference (RNAi) is a powerful tool that is being increasingly utilized for crop protection against viruses, fungal pathogens, and insect pests. The non-transgenic approach of spray-induced gene silencing (SIGS), which relies on spray application of double-stranded RNA (dsRNA) to induce RNAi, has come to prominence due to its safety and environmental benefits in addition to its wide host range and high target specificity. However, along with promising results in recent studies, several factors limiting SIGS RNAi efficiency have been recognized in insects and plants. While sprayed dsRNA on the plant surface can produce a robust RNAi response in some chewing insects, plant uptake and systemic movement of dsRNA is required for delivery to many other target organisms. For example, pests such as sucking insects require the presence of dsRNA in vascular tissues, while many fungal pathogens are predominately located in internal plant tissues. Investigating the mechanisms by which sprayed dsRNA enters and moves through plant tissues and understanding the barriers that may hinder this process are essential for developing efficient ways to deliver dsRNA into plant systems. In this review, we assess current knowledge of the plant foliar and cellular uptake of dsRNA molecules. We will also identify major barriers to uptake, including leaf morphological features as well as environmental factors, and address methods to overcome these barriers.

Keywords: RNAi; SIGS; double-stranded RNA; foliar dsRNA spray; nanoparticles; plant uptake of dsRNA.

Figure 1

Functional crop protection via a foliar dsRNA spray to induce RNAi. (A). Plant uptake of dsRNA can be divided into two stages: foliar uptake where sprayed dsRNA molecules from the leaf surfaces enter the interior of the leaf tissue, and cellular uptake where dsRNA molecules get taken up into plant cells. Following foliar uptake, sprayed dsRNAs may diffuse through the leaf interior and cellular uptake may occur. (B). Once dsRNA penetrates the cell wall pores and cell membrane to enter the cytoplasm, the plant RNAi machinery can process dsRNAs into siRNAs. Produced siRNAs can lead to degradation of viral transcripts in local cells and also be transported to adjacent cells. siRNAs are likely to participate in long distance signaling through vascular bundles to other parts of the plant. It is uncertain how non-processed dsRNA in the apoplastic pathway are translocated systemically. (C). dsRNA/siRNAs from the plant surfaces or in the plant system can be taken up by different targets and trigger an RNAi response depending on their sensitivity to dsRNA or siRNA. Figure created with BioRender.com.

Figure 2

Overview of barriers to efficient foliar and cellular uptake of dsRNAs. (A). Leaf wettability determined by trichomes, stomata, hydrophobic cuticle, and wax crystals acts as a barrier to foliar uptake of sprayed dsRNA. (B). Cell walls and cell membranes may hinder cellular uptake of dsRNA after sprayed dsRNA gets inside the leaf from the surface. (C). Environmental factors can contribute to degradation of dsRNA on the plant surfaces, thus acting as limiting factors to plant uptake of sufficient dsRNA. (D). Preferable procedures used to overcome these barriers and enhance uptake include the use of surfactants, high-pressure spray, and nanocarriers such as carbon dots, clay nanosheets, and single-walled carbon nanotubes. Figure created with BioRender.com.