Investigation of transitive and systemic RNA silencing in plants
Final Report Abstract
In this proposal, we investigated the parameters influencing the onset of transitive and systemic silencing in plants. Using a high-pressure spraying method, discrete in vitro synthesized RNA molecules could be delivered to Nicotiana benthamiana plants. We found that 22-nt small interfering RNAs (siRNAs) are the most potent inducers of transitive and systemic silencing of an intronless green fluorescent protein (GFP) transgene in N. benthamiana. However, an intron-containing GFP transgene was not prone to transitivity and systemic silencing, highlighting the important role of the introns in the onset of these processes. Intron-containing transcripts are processed by the spliceosome and may thereby escape RNA-directed RNA polymerase 6 (RDR6) processing. RDR6-mediated production of secondary dsRNA is an indispensable step for transitive and systemic silencing. Yet, we found that when intron-containing transgenes are not stably integrated but are transiently expressed as extrachromosomal entities, they could serve as RDR6 substrates. We propose that transcripts derived from extrachromosomal DNA are inefficiently spliced due to impaired spliceosome recruitment. The unspliced RNA appeared to serve as substrate of RDR6. This proposal was mainly based on delivery of RNA molecules in herbaceous plants by high pressure spraying. By pursuing the technical scope of this proposal, we developed trunk injection and petiole absorption methods to deliver RNA to woody plants. Our data demonstrated that RNA molecules introduced by trunk injection and petiole absorption are transported exclusively through the xylem. In the xylem and apoplast, they were quite stable and, importantly, they were not processed into siRNAs by DCLs. These findings were novel and of paramount importance for RNAi-based past management platforms.
Publications
- (2016) Induction of silencing in plants by high-pressure spraying of in vitro-synthesized small RNAs. Front Plant Sci, 7: 1327
Dalakouras, A., Wassenegger, M., McMillan, J., Cardoza, V., Maegele, I., Dadami, E., Runne, M., Krczal, G. and Wassenegger, M.
(See online at https://doi.org/10.3389/fpls.2016.01327) - (2018) Delivery of exogenous RNA molecules to woody and herbaceous plants by trunk injection and petiole absorption. Front Plant Sci, 9: 1253
Dalakouras, A., Jarausch, W., Buchholz, G., Bassler, A., Braun, M., Manthey, T., Krczal, G. and Wassenegger, M.
(See online at https://doi.org/10.3389/fpls.2018.01253) - (2018) Transient expression of intron ‑ containing transgenes generates non ‑ spliced aberrant pre‑mRNAs that are processed into siRNAs. Planta
Dalakouras, A., Lauter, A., Bassler, A., Krczal, G. and Wassenegger, M.
(See online at https://doi.org/10.1007/s00425-018-3015-6)
