Synthetic biology approach for plant protection using dsRNA

Summary

Pathogens 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 ds RNA quality, stability and efficient large‐scale production. Using components of the bacteriophage phi6, we engineered a stable and accurate in vivo ds RNA production system in Pseudomonas syringae bacteria. Unlike other in vitro or in vivo ds RNA production systems that rely on DNA transcription and postsynthetic alignment of single‐stranded RNA molecules, the phi6 system is based on the replication of ds RNA by an RNA‐dependent RNA polymerase, thus allowing production of high‐quality, long ds RNA molecules. The phi6 replication complex was reprogrammed to multiply ds RNA 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 ds RNA was achieved by combining all the three phi6 genome segments and by maintaining the natural ds RNA sizes and sequence elements required for efficient replication and packaging of the segments. The produced TMV‐derived ds RNAs inhibited TMV propagation when applied to infected Nicotiana benthamiana plants. The established ds RNA production system enables the broad application of ds RNA molecules as an efficient, highly flexible, nontransgenic and environmentally friendly approach for protecting crops against viruses and other pathogens.