Synthetic biology approach for plant protection using dsRNA

Abstract

SummaryPathogens induce severe damages on cultivated plants and represent a serious threat to global food security. Emerging strategies for crop protection involve the external treatment of plants with double‐stranded (ds)RNA to trigger RNA interference. However, applying this technology in greenhouses and fields depends on dsRNA quality, stability and efficient large‐scale production. Using components of the bacteriophage phi6, we engineered a stable and accurate in vivo dsRNA production system in Pseudomonas syringae bacteria. Unlike other in vitro or in vivo dsRNA production systems that rely on DNA transcription and postsynthetic alignment of single‐stranded RNA molecules, the phi6 system is based on the replication of dsRNA by an RNA‐dependent RNA polymerase, thus allowing production of high‐quality, long dsRNA molecules. The phi6 replication complex was reprogrammed to multiply dsRNA sequences homologous to tobacco mosaic virus (TMV) by replacing the coding regions within two of the three phi6 genome segments with TMV sequences and introduction of these constructs into P. syringae together with the third phi6 segment, which encodes the components of the phi6 replication complex. The stable production of TMV dsRNA was achieved by combining all the three phi6 genome segments and by maintaining the natural dsRNA sizes and sequence elements required for efficient replication and packaging of the segments. The produced TMV‐derived dsRNAs inhibited TMV propagation when applied to infected Nicotiana benthamiana plants. The established dsRNA production system enables the broad application of dsRNA molecules as an efficient, highly flexible, nontransgenic and environmentally friendly approach for protecting crops against viruses and other pathogens.

RNA-based disease control as a complementary measure to fight Fusarium fungi through silencing of the azole target Cytochrome P450 Lanosterol C-14 α-Demethylase

“…1b) has the potential to become an efficient disease control method. Direct application of dsRNAs or sRNAs onto host plants or post-harvest products circumvents transgenic approaches, leading to silencing of the target microbe/ pest gene and efficient disease control Koch et al 2016;Niehl et al 2018). Here we addressed the question whether Fusarium species other than Fg are sensitive to dsRNA derived from homologous fungal CYP51 genes.…”

Topically delivered 22 nt siRNAs enhance RNAi silencing of endogenous genes in two species

“…RNAi has been used to create a number of valuable traits in plants, and there is great potential for future applications to crop improvement (Frizzi and Huang 2010). Recently, plants have been shown to respond to RNA applied topically to leaf surfaces (Dalakouras et al 2016;Mitter et al 2017;Huang et al 2018;Niehl et al 2018;Sammons et al 2018;Dubrovina et al 2019), and this delivery route would expand the opportunities for using RNAi for agronomic benefit. In addition, topical delivery of sRNAs to plant cells provides a tool that can be used to investigate details of the RNAi mechanisms in plants.…”

Double-Stranded RNAs in Plant Protection Against Pathogenic Organisms and Viruses in Agriculture

“…Therefore, it has been proposed to use an inducible cassette with the T7 phage RNA polymerase promoter, which expresses dsRNA in the RNase III-deficient Escherichia coli strain HT115, M-JM109, or M-JM109lacY [78][79][80][81]. In addition, stable and efficient systems for dsRNA production in Pseudomonas syringae bacteria [82] and Saccharomyces cerevisiae yeast [83] were recently developed. Obviously, the listed microbiological expression systems can potentially be used for large-scale and inexpensive production of dsRNAs for practical applications of SIGS in agriculture.…”