An optimal RNAi configuration that could restrict gene expression most efficiently was determined. This approach was also used to target PTGS and yielded higher rates of gene-editing events.
Although it was characterized long ago, transgene silencing still strongly impairs transgene overexpression, and thus is a major barrier to plant crop gene-editing. The development of strategies that could prevent transgene silencing is therefore essential to the success of gene editing assays. Transgene silencing occurs via the RNA silencing process, which regulates the expression of essential genes and protects the plant from viral infections. The RNA silencing machinery thereby controls central biological processes such as growth, development, genome integrity, and stress resistance. RNA silencing is typically induced by aberrant RNA, that may lack 5′ or 3′ processing, or may consist in double-stranded or hairpin RNA, and involves DICER and ARGONAUTE family proteins. In this study, RNAi inducing constructs were designed in eleven different configurations and were evaluated for their capacity to induce silencing in Nicotiana spp. using transient and stable transformation assays. Using reporter genes, it was found that the overexpression of a hairpin consisting of a forward tandem inverted repeat that started with an ATG and that was not followed downstream by a transcription terminator, could downregulate gene expression most potently. Furthermore, using this method, the downregulation of the NtSGS3 gene caused a significant increase in transgene expression both in transient and stable transformation assays. This SGS3 silencing approach was also employed in gene-editing assays and caused higher rates of gene-editing events. Taken together, these findings suggested the optimal genetic configuration to cause RNA silencing and showed that this strategy may be used to restrict PTGS during gene-editing experiments.
This is a preview of subscription content, access via your institution.
Buy single article
Instant access to the full article PDF.
Price excludes VAT (USA)
Tax calculation will be finalised during checkout.
The data underlying this article are available in the article and in its online supplemental material.
Ali S, Kim W-C (2019) A fruitful decade using synthetic promoters in the improvement of transgenic plants. Front Plant Sci 10:1433
Assaad FF, Tucker KL, Signer ER (1993) Epigenetic repeat-induced gene silencing (RIGS) in Arabidopsis. Plant Mol Biol 22:1067–1085
Bartel DP (2004) MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 116:281–297
Baulcombe DC (1996) RNA as a target and an initiator of post-transcriptional gene silencing in transgenic plants. Plant Mol Biol 32:79–88
Baulcombe D (2004) RNA silencing in plants. Nature 431:356–363
Baulcombe DC (2007) Molecular biology. Amplified silencing. Science 315:199–200
Benhabiles H, Jia J, Lejeune F (2016) Chapter 1—general aspects related to nonsense mutations. In: Benhabiles H, Jia J, Lejeune F (eds) Nonsense mutation correction in human diseases. Academic Press, Boston, pp 1–76
Brosnan CA, Mitter N, Christie M et al (2007) Nuclear gene silencing directs reception of long-distance mRNA silencing in Arabidopsis. Proc Natl Acad Sci USA 104:14741–14746
Butaye KMJ, Goderis IJWM, Wouters PFJ et al (2004) Stable high-level transgene expression in Arabidopsis thaliana using gene silencing mutants and matrix attachment regions. Plant J 39:440–449
Chuang CF, Meyerowitz EM (2000) Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proc Natl Acad Sci USA 97:4985–4990
Czarnecki O, Bryan AC, Jawdy SS et al (2016) Simultaneous knockdown of six non-family genes using a single synthetic RNAi fragment in Arabidopsis thaliana. Plant Methods 12:16
Dafny-Yelin M, Chung S-M, Frankman EL, Tzfira T (2007) pSAT RNA interference vectors: a modular series for multiple gene down-regulation in plants. Plant Physiol 145:1272–1281
Ding S-W, Voinnet O (2007) Antiviral immunity directed by small RNAs. Cell 130:413–426
Doyle J (1991) DNA protocols for plants. In: Hewitt GM, Johnston AWB, Young JPW (eds) Molecular techniques in taxonomy. Springer, Berlin, pp 283–293
Elmayan T, Adenot X, Gissot L et al (2009) A neomorphic sgs3 allele stabilizing miRNA cleavage products reveals that SGS3 acts as a homodimer. FEBS J 276:835–844
Fagard M, Vaucheret H (2000) (TRANS)Gene silencing in plants: how many mechanisms? Annu Rev Plant Physiol Plant Mol Biol 51:167–194
Havecker ER, Wallbridge LM, Hardcastle TJ et al (2010) The Arabidopsis RNA-directed DNA methylation argonautes functionally diverge based on their expression and interaction with target loci. Plant Cell 22:321–334
Helliwell CA, Varsha Wesley S, Wielopolska AJ, Waterhouse PM (2002) High-throughput vectors for efficient gene silencing in plants. Funct Plant Biol 29:1217–1225
Höfgen R, Willmitzer L (1988) Storage of competent cells for Agrobacterium transformation. Nucleic Acids Res 16:9877
Hsu C-T, Lee W-C, Cheng Y-J et al (2020) Genome editing and protoplast regeneration to study plant-pathogen interactions in the model plant Nicotiana benthamiana. Front Genome Ed 2:627803
Jiang P, Zhang K, Ding Z et al (2018) Characterization of a strong and constitutive promoter from the Arabidopsis serine carboxypeptidase-like gene AtSCPL30 as a potential tool for crop transgenic breeding. BMC Biotechnol 18:59
Johansen LK, Carrington JC (2001) Silencing on the spot. Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system. Plant Physiol 126:930–938
Kloc A, Martienssen R (2008) RNAi, heterochromatin and the cell cycle. Trends Genet 24:511–517
Kumar M, Ayzenshtat D, Marko A, Bocobza S (2021) Optimization of T-DNA configuration with UBIQUITIN10 promoters and tRNA-sgRNA complexes promotes highly efficient genome editing in allotetraploid tobacco. Plant Cell Rep. https://doi.org/10.1007/s00299-021-02796-0
Law JA, Jacobsen SE (2010) Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nat Rev Genet 11:204–220
Li X, Jiang D-H, Yong K, Zhang D-B (2007) Varied transcriptional efficiencies of multiple Arabidopsis U6 small nuclear RNA genes. J Integr Plant Biol 49:222–229
Lin C-S, Hsu C-T, Yuan Y-H et al (2022) DNA-free CRISPR-Cas9 gene editing of wild tetraploid tomato Solanum peruvianum using protoplast regeneration. Plant Physiol 188:1917–1930
Lippman Z, Martienssen R (2004) The role of RNA interference in heterochromatic silencing. Nature 431:364–370
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408
Luo Z, Chen Z (2007) Improperly terminated, unpolyadenylated mRNA of sense transgenes is targeted by RDR6-mediated RNA silencing in Arabidopsis. Plant Cell 19:943–958
Mallory AC, Vaucheret H (2006) Functions of microRNAs and related small RNAs in plants. Nat Genet 38(Suppl):S31–S36
Marjanac G, Karimi M, Naudts M et al (2009) Gene silencing induced by hairpin or inverted repeated sense transgenes varies among promoters and cell types. New Phytol 184:851–864
Matsuo K, Atsumi G (2019) CRISPR/Cas9-mediated knockout of the RDR6 gene in Nicotiana benthamiana for efficient transient expression of recombinant proteins. Planta 250:463–473
Mlotshwa S, Pruss GJ, Peragine A et al (2008) DICER-LIKE2 plays a primary role in transitive silencing of transgenes in Arabidopsis. PLoS ONE 3:e1755
Moissiard G, Parizotto EA, Himber C, Voinnet O (2007) Transitivity in Arabidopsis can be primed, requires the redundant action of the antiviral Dicer-like 4 and Dicer-like 2, and is compromised by viral-encoded suppressor proteins. RNA 13:1268–1278
Mourrain P, Béclin C, Elmayan T et al (2000) Arabidopsis SGS2 and SGS3 genes are required for posttranscriptional gene silencing and natural virus resistance. Cell 101:533–542
Norris SR, Meyer SE, Callis J (1993) The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression. Plant Mol Biol 21:895–906
Pak J, Fire A (2007) Distinct populations of primary and secondary effectors during RNAi in C. elegans. Science 315:241–244
Palauqui JC, Elmayan T, Pollien JM, Vaucheret H (1997) Systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions. EMBO J 16:4738–4745
Parent J-S, Jauvion V, Bouché N et al (2015) Post-transcriptional gene silencing triggered by sense transgenes involves uncapped antisense RNA and differs from silencing intentionally triggered by antisense transgenes. Nucleic Acids Res 43:8464–8475
Peragine A, Yoshikawa M, Wu G et al (2004) SGS3 and SGS2/SDE1/RDR6 are required for juvenile development and the production of trans-acting siRNAs in Arabidopsis. Genes Dev 18:2368–2379
Peters L, Meister G (2007) Argonaute proteins: mediators of RNA silencing. Mol Cell 26:611–623
Polturak G, Breitel D, Grossman N et al (2016) Elucidation of the first committed step in betalain biosynthesis enables the heterologous engineering of betalain pigments in plants. New Phytol 210:269–283
Sarrion-Perdigones A, Vazquez-Vilar M, Palací J et al (2013) GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol 162:1618–1631
Schwach F, Vaistij FE, Jones L, Baulcombe DC (2005) An RNA-dependent RNA polymerase prevents meristem invasion by potato virus X and is required for the activity but not the production of a systemic silencing signal. Plant Physiol 138:1842–1852
Schweizer P, Pokorny J, Schulze-Lefert P, Dudler R (2000) Technical advance. Double-stranded RNA interferes with gene function at the single-cell level in cereals. Plant J 24:895–903
Sijen T, Fleenor J, Simmer F et al (2001) On the role of RNA amplification in dsRNA-triggered gene silencing. Cell 107:465–476
Smith NA, Singh SP, Wang MB et al (2000) Total silencing by intron-spliced hairpin RNAs. Nature 407:319–320
Sparkes IA, Runions J, Kearns A, Hawes C (2006) Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc 1:2019–2025
Stoutjesdijk PA, Singh SP, Liu Q et al (2002) hpRNA-mediated targeting of the Arabidopsis FAD2 gene gives highly efficient and stable silencing. Plant Physiol 129:1723–1731
Strack D, Vogt T, Schliemann W (2003) Recent advances in betalain research. Phytochemistry 62:247–269
Timoneda A, Feng T, Sheehan H et al (2019) The evolution of betalain biosynthesis in Caryophyllales. New Phytol 224:71–85
Vaistij FE, Jones L, Baulcombe DC (2002) Spreading of RNA targeting and DNA methylation in RNA silencing requires transcription of the target gene and a putative RNA-dependent RNA polymerase. Plant Cell 14:857–867
van der Krol AR, Mur LA, Beld M et al (1990) Flavonoid genes in petunia: addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 2:291–299
Vaucheret H (2008) Plant Argonautes. Trends Plant Sci 13:350–358
Vaucheret H, Béclin C, Fagard M (2001) Post-transcriptional gene silencing in plants. J Cell Sci 114:3083–3091
Venkataraman S, Prasad BVLS, Selvarajan R (2018) RNA dependent RNA Polymerases: insights from structure, function and evolution. Viruses. https://doi.org/10.3390/v10020076
Waterhouse PM, Graham MW, Wang MB (1998) Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci USA 95:13959–13964
Weinhold A, Kallenbach M, Baldwin IT (2013) Progressive 35S promoter methylation increases rapidly during vegetative development in transgenic Nicotiana attenuata plants. BMC Plant Biol 13:99
Wesley SV, Helliwell CA, Smith NA et al (2001) Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J 27:581–590
Xie Z, Johansen LK, Gustafson AM et al (2004) Genetic and functional diversification of small RNA pathways in plants. PLoS Biol 2:E104
Yan H, Chretien R, Ye J, Rommens CM (2006) New construct approaches for efficient gene silencing in plants. Plant Physiol 141:1508–1518
Yan P, Shen W, Gao X et al (2012) High-throughput construction of intron-containing hairpin RNA vectors for RNAi in plants. PLoS One 7:e38186
Yifhar T, Pekker I, Peled D et al (2012) Failure of the tomato trans-acting short interfering RNA program to regulate AUXIN RESPONSE FACTOR3 and ARF4 underlies the wiry leaf syndrome. Plant Cell 24:3575–3589
Yoshikawa M, Peragine A, Park MY, Poethig RS (2005) A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes Dev 19:2164–2175
Yoshikawa M, Iki T, Tsutsui Y et al (2013) 3’ fragment of miR173-programmed RISC-cleaved RNA is protected from degradation in a complex with RISC and SGS3. Proc Natl Acad Sci USA 110:4117–4122
Yu D, Liao L, Zhang Y et al (2018) Development of a Gateway-compatible pCAMBIA binary vector for RNAi-mediated gene knockdown in plants. Plasmid 98:52–55
Zemach A, Kim MY, Hsieh P-H et al (2013) The Arabidopsis nucleosome remodeler DDM1 allows DNA methyltransferases to access H1-containing heterochromatin. Cell 153:193–205
Zhang Z, Liu X, Guo X et al (2016) Arabidopsis AGO3 predominantly recruits 24-nt small RNAs to regulate epigenetic silencing. Nat Plants 2:16049
We are very thankful to Dr. Tzahi Arazi for his precious advice and critical reading of this manuscript. We are also grateful to Prof. Asaph Aharoni for providing the pX11 construct (Polturak et al. 2016) used for betalain biosynthesis.
This study was financially supported by the Chief Scientist—Ministry of Agriculture and Rural Development No. 20-01-0209 as part of the National Center for Genome Editing in Agriculture.
Conflict of interest
The authors declare that they have no conflicts of interest.
Communicated by Baochun Li.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Kumar, M., Tripathi, P.K., Ayzenshtat, D. et al. Increased rates of gene-editing events using a simplified RNAi configuration designed to reduce gene silencing. Plant Cell Rep 41, 1987–2003 (2022). https://doi.org/10.1007/s00299-022-02903-9
- Gene silencing
- Plant transformation
- Gene editing