Artificial Small RNAs for Functional Genomics in Plants

Cisneros, Adriana E; Torre-Montaña, Ainhoa de la; Martín-García, Tamara; Carbonell, Alberto

doi:10.1007/978-3-030-64994-4_1

Cited by 3 publications

(1 citation statement)

References 159 publications

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“…For both art‐sRNA classes, the guide strand of the art‐sRNA duplex (typically including a 5′ U) associates with AGO1 to silence complementary RNA(s). The art‐sRNAs are extensively used in plants to regulate gene expression in gene function studies and to induce antiviral resistance (Cisneros & Carbonell, 2020; Cisneros et al., 2021)]. However, one major limitation of current art‐sRNA approaches is that art‐sRNA are usually expressed in transgenic organisms, which hinders the commercialization of art‐sRNA‐expressing crops in regions where genetically modified organisms (GMOs) are banned.…”

Section: Introductionmentioning

confidence: 99%

Systemic silencing of an endogenous plant gene by two classes of mobile 21‐nucleotide artificial small RNAs

2022

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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 21nucleotide 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.

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Section: Introductionmentioning

confidence: 99%

Systemic silencing of an endogenous plant gene by two classes of mobile 21‐nucleotide artificial small RNAs

2022

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Artificial miRNAs: A potential tool for genetic improvement of horticultural crops

Kumar,

Panwar,

Chaudhary

et al. 2024

Scientia Horticulturae

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Syn-tasiR-VIGS: Virus-Based Targeted RNAi in Plants By Synthetic Trans Acting Small Interfering RNAs Derived from Minimal Precursors

Cisneros,

Alarcia,

Llorens-Gámez

et al. 2024

Preprint

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Synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are 21-nucleotide (nt) small RNAs designed to silence plant transcripts with high specificity. Their use as biotechnological tools for functional genomics and crop improvement is limited by the need to transgenically express longTASprecursors to produce syn-tasiRNAsin vivo. Here, we show that authentic and highly effective syn-tasiRNAs can be produced from minimal, non-TASprecursors consisting of a 22-nt endogenous microRNA target site, an 11-nt spacer and the 21 nt syn-tasiRNA sequence(s). These minimal precursors, when transgenically expressed inArabidopsis thalianaandNicotiana benthamiana, generated highly phased syn-tasiRNAs that silenced one or multiple plant genes with high efficacy. Remarkably, minimal but not full-lengthTASprecursors produced authentic syn-tasiRNAs and induced widespread gene silencing inN. benthamianawhen expressed from an RNA virus, which can be applied by spraying infectious crude extracts onto leaves in a GMO-free manner. This strategy, named syn-tasiRNA-based virus-induced gene silencing (syn-tasiR-VIGS), was further used to vaccinate plants against a pathogenic virus, resulting in complete plant immunization. Our results reveal that syn-tasiRNA precursors can be significantly shortened without compromising silencing efficacy, and that syn-tasiR-VIGS represents a versatile, scalable and non-transgenic platform for precision RNAi and antiviral vaccination in plants.Graphical abstract

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Artificial Small RNAs for Functional Genomics in Plants

Cited by 3 publications

References 159 publications

Systemic silencing of an endogenous plant gene by two classes of mobile 21‐nucleotide artificial small RNAs

Systemic silencing of an endogenous plant gene by two classes of mobile 21‐nucleotide artificial small RNAs

Artificial miRNAs: A potential tool for genetic improvement of horticultural crops

Syn-tasiR-VIGS: Virus-Based Targeted RNAi in Plants By Synthetic Trans Acting Small Interfering RNAs Derived from Minimal Precursors

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