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Fine-tune control of targeted RNAi efficacy by plant artificial small RNAs.

ABSTRACT: Eukaryotic RNA interference (RNAi) results in gene silencing upon the sequence-specific degradation of target transcripts by complementary small RNAs (sRNAs). In plants, RNAi-based tools have been optimized for high efficacy and high specificity, and are extensively used in gene function studies and for crop improvement. However, efficient methods for finely adjusting the degree of induced silencing are missing. Here, we present two different strategies based on artificial sRNAs for fine-tuning targeted RNAi efficacy in plants. First, the degree of silencing induced by synthetic-trans-acting small interfering RNAs (syn-tasiRNAs) can be adjusted by modifying the precursor position from which the syn-tasiRNA is expressed. The accumulation and efficacy of Arabidopsis TAS1c-based syn-tasiRNAs progressively decrease as the syn-tasiRNA is expressed from positions more distal to the trigger miR173 target site. And second, syn-tasiRNA activity can also be tweaked by modifying the degree of base-pairing between the 3' end of the syn-tasiRNA and the 5' end of the target RNA. Both strategies were used to finely modulate the degree of silencing of endogenous and exogenous target genes in Arabidopsis thaliana and Nicotiana benthamiana. New high-throughput syn-tasiRNA vectors were developed and functionally analyzed, and should facilitate the precise control of gene expression in multiple plant species.

SUBMITTER: Lopez-Dolz L

PROVIDER: S-EPMC7293048 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Fine-tune control of targeted RNAi efficacy by plant artificial small RNAs.

López-Dolz Lucio L Spada Maria M Daròs José-Antonio JA Carbonell Alberto A

Nucleic acids research 20200601 11

Eukaryotic RNA interference (RNAi) results in gene silencing upon the sequence-specific degradation of target transcripts by complementary small RNAs (sRNAs). In plants, RNAi-based tools have been optimized for high efficacy and high specificity, and are extensively used in gene function studies and for crop improvement. However, efficient methods for finely adjusting the degree of induced silencing are missing. Here, we present two different strategies based on artificial sRNAs for fine-tuning ...[more]

PMID: 32396204

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Project description:BackgroundRNA silencing is an important mechanism for regulation of endogenous gene expression and defense against genomic intruders in plants. This natural defense system was adopted to generate virus-resistant plants even before the mechanism of RNA silencing was unveiled. With the clarification of that mechanism, transgenic antiviral plants were developed that expressed artificial virus-specific hairpin RNAs (hpRNAs) or microRNAs (amiRNAs) in host plants. Previous works also showed that plant-mediated RNA silencing technology could be a practical method for constructing insect-resistant plants by expressing hpRNAs targeting essential genes of insects.Methodology/principal findingsIn this study, we chose aphid Myzus persicae of order Hemiptera as a target insect. To screen for aphid genes vulnerable to attack by plant-mediated RNA silencing to establish plant aphid resistance, we selected nine genes of M. persicae as silencing targets, and constructed their hpRNA-expressing vectors. For the acetylcholinesterase 2 coding gene (MpAChE2), two amiRNA-expressing vectors were also constructed. The vectors were transformed into tobacco plants (Nicotiana tabacum cv. Xanti). Insect challenge assays showed that most of the transgenic plants gained aphid resistance, among which those expressing hpRNAs targeting V-type proton ATPase subunit E-like (V-ATPaseE) or tubulin folding cofactor D (TBCD) genes displayed stronger aphicidal activity. The transgenic plants expressing amiRNAs targeting two different sites in the MpAChE2 gene exhibited better aphid resistance than the plants expressing MpAChE2-specific hpRNA.Conclusions/significanceOur results indicated that plant-mediated insect-RNA silencing might be an effective way to develop plants resistant to insects with piercing-sucking mouthparts, and both the selection of vulnerable target genes and the biogenetic type of the small RNAs were crucial for the effectiveness of aphid control. The expression of insect-specific amiRNA is a promising and preferable approach to engineer plants resistant to aphids and, possibly, to other plant-infesting insects.

Project description:Artificial small RNAs (art-sRNAs) are 21-nucleotide small RNAs (sRNAs) computationally designed to silence plant genes or pathogenic RNAs with high efficacy and specificity. They are typically produced in transgenic plants to induce silencing at the whole-organism level, although their expression in selected tissues for inactivating genes in distal tissues has not been reported. Here, art-sRNAs designed against the magnesium chelatase subunit CHLI-encoding SULFUR gene (NbSu) were agroinfiltrated in Nicotiana benthamiana leaves, and the induction of local and systemic silencing was analyzed phenotypically by monitoring the appearance of the characteristic bleached phenotype, as well as molecularly by analyzing art-sRNA processing, accumulation and targeting activity and efficacy. We found that the two classes of art-sRNAs, artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs), are able to induce systemic silencing of NbSu, which requires high art-sRNA expression in the vicinity of the leaf petiole but is independent on the production of secondary sRNAs from NbSu mRNAs. Moreover, we revealed that 21-nucleotide amiRNA and syn-tasiRNA duplexes, and not their precursors, are the molecules moving between cells and through the phloem to systemically silence NbSu in upper leaves. In sum, our results indicate that 21-nucleotide art-sRNAs can move throughout the plant to silence plant genes in tissues different from where they are produced. This highlights the biotechnological potential of art-sRNAs, which might be applied locally for triggering whole-plant and highly specific silencing to regulate gene expression or induce resistance against pathogenic RNAs in next-generation crops. The present study demonstrates that artificial small RNAs, such as artificial microRNAs and synthetic trans-acting small interfering RNAs, can move long distances in plants as 21-nucleotide duplexes, specifically silencing endogenous genes in tissues different from where they are applied. This highlights the biotechnological potential of artificial small RNAs, which might be applied locally for triggering whole-plant, highly specific silencing to regulate gene expression or induce resistance against pathogenic RNAs in next-generation crops.

Dataset Information

Fine-tune control of targeted RNAi efficacy by plant artificial small RNAs.

Publications

Fine-tune control of targeted RNAi efficacy by plant artificial small RNAs.

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