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.
Highlights
Agricultural yields are greatly affected by various pathogens and pests which decrease production of crops worldwide (Oerke, 2005) with losses adding up to around $US100 billion per year (Fletcher et al, 2006) and potentially reaching U$540 billion per year if the spreading of invasive pests and pathogens is not controlled (Kew Royal Botanical Gardens, 2017; Paini et al, 2016)
The dsRNAs were applied to Nicotiana benthamiana plants infected with a GFP-tagged tobacco mosaic virus (TMV-GFP) which allows the in vivo monitoring of infection by the analysis of GFP fluorescence
The polymerase complex (PC) that forms the internal layer of the phi6 phage particle is delivered to the cytoplasm of the host bacterium
Summary
Agricultural yields are greatly affected by various pathogens and pests which decrease production of crops worldwide (Oerke, 2005) with losses adding up to around $US100 billion per year (Fletcher et al, 2006) and potentially reaching U$540 billion per year if the spreading of invasive pests and pathogens is not controlled (Kew Royal Botanical Gardens, 2017; Paini et al, 2016). To protect plants against pathogens and pests, agriculture relies on the widespread use of chemical pesticides that are applied to the environment in large amounts. These intense applications of chemical pesticides pose potential risks of human health, beneficial organisms and the environment (NicolopoulouStamati et al, 2016). Legislation in Europe is aiming at reducing the use of chemical plant protection products and calling for an in-depth reconsideration of crop protection solutions (Union, 2009). A promising new approach with strong potential for protecting plants against viruses and other pathogens involves the application of doublestranded (ds)RNA
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
