In a recent groundbreaking study conducted by a research team at Tokyo University of Agriculture and Technology, it has been discovered that long double-stranded RNAs (dsRNAs) efficiently induce RNA interference (RNAi) in plant cells, a finding that holds significant potential for agricultural biotechnology and crop improvement. Published in the esteemed journal Scientific Reports on May 6, 2019, and identifiable via the Digital Object Identifier (DOI) 10.1038/s41598-019-43443-9, this research has revolutionized our understanding of RNAi and its implications for plant biology and disease resistance.
The research was spearheaded by Sayaka S. Kakiyama, Midori M. Tabara, Yuki Y. Nishibori, Hiromitsu H. Moriyama, and Toshiyuki T. Fukuhara from the Department of Applied Biological Sciences at the Tokyo University of Agriculture and Technology. With funding from non-U.S. governmental sources, the group set out to explore the mechanics of RNAi, a biological process in which RNA molecules inhibit gene expression or translation, effectively silencing targeted genes.
RNA interference has been a subject of intense research ever since its first discovery in the petunia plants (Napoli et al., 1990, doi: 10.1105/tpc.2.4.279) and subsequently in the animal model organism Caenorhabditis elegans by Fire et al. (Nature, 1998, doi: 10.1038/35888). Moreover, the work of Bernstein et al. (Nature, 2001, doi: 10.1038/35053110) and the collective understanding of the RNAi mechanism have been instrumental in developing gene-silencing technologies that are used both in basic research and potential therapies.
The Japanese team’s research now adds a new chapter to this story, comparing the efficacy of various sizes of dsRNAs from 21 to 139 nucleotides (nt) in inducing RNAi via the direct transfer of these dsRNAs into protoplasts prepared from Arabidopsis thaliana seedlings. A protoplast is a plant cell with its cell wall removed, allowing the entry of foreign molecules such as dsRNAs.
The experimental findings revealed that Dicer-like 4 (DCL4), a key enzyme in the process, preferentially cleaves long dsRNAs in the protoplasts. Previous biochemical data supported this preference, suggesting that DCL4 plays a crucial role in the RNAi mechanism. The study showed that when long dsRNAs of approximately 130 nt were introduced directly into protoplasts, they induced RNAi far more effectively — by approximately 60 to 400-fold — than shorter 37-nt dsRNAs.
Interestingly, while the transfer of 21-nt dsRNAs did trigger RNAi without DCL4 activity, the process was much less efficient. The findings imply that for a potent RNAi response, the cleavage of long dsRNAs into 21-nt segments by DCL4 is essential. This breakthrough in understanding how RNAi functions in plant cells may herald significant applications in agriculture, such as developing resistance to pests and diseases without the use of chemicals.
The study’s format as a direct transfer of dsRNAs into Arabidopsis protoplasts suggests a versatile methodology that could be adapted for various agricultural applications. Their findings resonate with the observations of Fagard et al. (Proc. Natl. Acad. Sci. USA, 2000, doi: 10.1073/pnas.200217597) and Hamilton and Baulcombe (Science, 1999, doi: 10.1126/science.286.5441.950) on post-transcriptional gene silencing.
Moreover, the team’s approach aligns with the innovative applications proposed by researchers such as Elbashir et al. (Nature, 2001, doi: 10.1038/35078107) and Kim et al. (Nat. Biotechnol., 2005, doi: 10.1038/nbt1051) who looked at RNAi as a robust tool for gene function studies and therapeutics in mammalian systems. By tweaking dsRNA length, one could potentially fine-tune the silencing of harmful genes in crops targeted by pathogens or pests, creating a natural defense mechanism encoded within the plants themselves.
There’s great hope for this technique as it allows for precise targeting without affecting the plant’s overall growth and development. As pointed out by Wesley et al. (Plant J., 2001, doi: 10.1046/j.1365-313X.2001.01105.x), the strategic design of RNA silencing constructs is critical for the efficient and effective gene silencing in plants.
The research implications extend beyond biosafety and agricultural sustainability, touching upon broader concerns about food security and environmental protection. In an era where climate change threatens crop yields and population growth demands increased food production, the study by Kakiyama et al. offers a glimpse of the innovative solutions that biotechnology can provide.
Keywords
1. RNA interference crops
2. dsRNA-induced gene silencing
3. Plant RNAi technology
4. Dicer-like proteins in plants
5. Protoplast transformation technique
Overall, the discovery opens up new avenues for the use of RNAi in plant genetics and crop improvement strategies. This research represents a significant step forward in biotechnology, offering a promising perspective on the future of sustainable agriculture.
References
1. Napoli, C., Lemieux, C., & Jorgensen, R. (1990). Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. The Plant Cell, 2(4), 279–289. doi: 10.1105/tpc.2.4.279.
2. Fire, A., et al. (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391, 806–811. doi: 10.1038/35888.
3. Bernstein, E., et al. (2001). Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature, 409, 363–366. doi: 10.1038/35053110.
4. Elbashir, S. M., et al. (2001). Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 411, 494–498. doi: 10.1038/35078107.
5. Wesley, S. V., et al. (2001). Construct design for efficient, effective and high-throughput gene silencing in plants. The Plant Journal, 27(6), 581–590. doi: 10.1046/j.1365-313X.2001.01105.x.
For further exploration and understanding of the study, please refer to the DOI link provided: https://doi.org/10.1038/s41598-019-43443-9.