Expression and processing of polycistronic artificial microRNAs and trans‐acting siRNAs from transiently introduced transgenes in Solanum lycopersicum and Nicotiana benthamiana

• Published in: The Plant journal : for cell and molecular biology 2021-05, Vol.106 (4), p.1087-1104
• Main Authors: Lunardon, Alice, Kariuki, Samwel Muiruri, Axtell, Michael J.
• Format: Article
• Language: English
• Subjects: Agroinfiltration; Gene sequencing; Gene Silencing; microRNA; MicroRNAs; MicroRNAs - genetics; miRNA; Multiplexing; Nicotiana - genetics; Nicotiana benthamiana; polycistronic; Polymerase chain reaction; Precursors; Ribonucleic acid; RNA; RNA processing; RNA Processing, Post-Transcriptional - genetics; RNA, Plant - genetics; RNA, Small Interfering - genetics; siRNA; Solanum lycopersicum; Solanum lycopersicum - genetics; Tomatoes; Transgenes
• ISSN: 0960-7412; 1365-313X
• DOI: 10.1111/tpj.15221

SUMMARY Targeted gene silencing using small regulatory RNAs is a widely used technique for genetic studies in plants. Artificial microRNAs are one common approach, as they have the advantage of producing just a single functional small RNA, which can be designed for high target specificity and low off‐target effects. Simultaneous silencing of multiple targets with artificial microRNAs can be achieved by producing polycistronic microRNA precursors. Alternatively, specialized trans‐acting short interfering RNA (tasiRNA) precursors can be designed to produce several specific tasiRNAs at once. Here we tested several artificial microRNA‐ and tasiRNA‐based methods for multiplexed gene silencing in Solanum lycopersicum (tomato) and Nicotiana benthamiana. All analyses used transiently expressed transgenes delivered by infiltration of leaves with Agrobacterium tumefacians. Small RNA sequencing analyses revealed that many previously described approaches resulted in poor small RNA processing. The 5′‐most microRNA precursor hairpins on polycistronic artificial microRNA precursors were generally processed more accurately than precursors at the 3′‐end. Polycistronic artificial microRNAs where the hairpin precursors were separated by transfer RNAs had the best processing precision. Strikingly, artificial tasiRNA precursors failed to be processed in the expected phased manner in our system. These results highlight the need for further development of multiplexed artificial microRNA and tasiRNA strategies. The importance of small RNA sequencing, as opposed to single‐target assays such as RNA blots or real‐time polymerase chain reaction, is also discussed. Significance Statement Several strategies for multiplexed gene silencing using artificial microRNAs or tasiRNAs have been described. We find that many result in imprecise processing, and thus low accumulation of the intended small RNAs. Our findings highlight the importance of small RNA sequencing to analyze gene silencing experiments fully, and the need for continued methodological development of these methods.