Transgene-free, virus-based gene silencing in plants by artificial microRNAs derived from minimal precursors.

Artificial microRNAs (amiRNAs) are highly specific, 21-nucleotide (nt) small RNAs designed to silence target transcripts. In plants, their application as biotechnological tools for functional genomics or crop improvement is limited by the need of transgenically expressing long primary miRNA (pri-miRNA) precursors to produce the amiRNAs in vivo. Here, we analyzed the minimal structural and sequence requirements for producing effective amiRNAs from the widely used, 521-nt long AtMIR390a pri-miRNA from Arabidopsis thaliana. We functionally screened in Nicotiana benthamiana a large collection of constructs transiently expressing amiRNAs against endogenous genes and from artificially shortened MIR390-based precursors and concluded that highly effective and accurately processed amiRNAs can be produced from a chimeric precursor of only 89 nt. This minimal precursor was further validated in A. thaliana transgenic plants expressing amiRNAs against endogenous genes. Remarkably, minimal but not full-length precursors produce authentic amiRNAs and induce widespread gene silencing in N. benthamiana when expressed from an RNA virus, which can be applied into leaves by spraying infectious crude extracts. Our results reveal that the length of amiRNA precursors can be shortened without affecting silencing efficacy, and that viral vectors including minimal amiRNA precursors can be applied in a transgene-free manner to induce whole-plant gene silencing.

Role of RNA silencing in plant-viroid interactions and in viroid pathogenesis.

Viroids are small, single-stranded, non-protein coding and circular RNAs able to infect host plants in the absence of any helper virus. They may elicit symptoms in their hosts, but the underlying molecular pathways are only partially known. Here we address the role of post-transcriptional RNA silencing in plant-viroid-interplay, with major emphasis on the involvement of this sequence-specific RNA degradation mechanism in both plant antiviroid defence and viroid pathogenesis. This review is a tribute to the memory of Dr. Ricardo Flores, who largely contributed to elucidate this and other molecular mechanisms involved in plant-viroid interactions.

Down-regulation of tomato STEROL GLYCOSYLTRANSFERASE 1 perturbs plant development and facilitates viroid infection.

Potato spindle tuber viroid (PSTVd) is a plant pathogen naturally infecting economically important crops such as tomato (Solanum lycopersicum). Here, we aimed to engineer tomato plants highly resistant to PSTVd and developed several S. lycopersicum lines expressing an artificial microRNA (amiRNA) against PSTVd (amiR-PSTVd). Infectivity assays revealed that amiR-PSTVd-expressing lines were not resistant but instead hypersusceptible to the viroid. A combination of phenotypic, molecular, and metabolic analyses of amiRNA-expressing lines non-inoculated with the viroid revealed that amiR-PSTVd was accidentally silencing the tomato STEROL GLYCOSYLTRANSFERASE 1 (SlSGT1) gene, which caused late developmental and reproductive defects such as leaf epinasty, dwarfism, or reduced fruit size. Importantly, two independent transgenic tomato lines each expressing a different amiRNA specifically designed to target SlSGT1 were also hypersusceptible to PSTVd, thus demonstrating that down-regulation of SlSGT1 was responsible for the viroid-hypersusceptibility phenotype. Our results highlight the role of sterol glycosyltransferases in proper plant development and indicate that the imbalance of sterol glycosylation levels favors viroid infection, most likely by facilitating viroid movement.

Diagnostics of Infections Produced by the Plant Viruses TMV, TEV, and PVX with CRISPR-Cas12 and CRISPR-Cas13.

Viral infections in plants threaten food security. Thus, simple and effective methods for virus detection are required to adopt early measures that can prevent virus spread. However, current methods based on the amplification of the viral genome by polymerase chain reaction (PCR) require laboratory conditions. Here, we exploited the CRISPR-Cas12a and CRISPR-Cas13a/d systems to detect three RNA viruses, namely, Tobacco mosaic virus, Tobacco etch virus, and Potato virus X, in Nicotiana benthamiana plants. We applied the CRISPR-Cas12a system to detect viral DNA amplicons generated by PCR or isothermal amplification, and we also performed a multiplexed detection in plants with mixed infections. In addition, we adapted the detection system to bypass the costly RNA purification step and to get a visible readout with lateral flow strips. Finally, we applied the CRISPR-Cas13a/d system to directly detect viral RNA, thereby avoiding the necessity of a preamplification step and obtaining a readout that scales with the viral load. These approaches allow for the performance of viral diagnostics within half an hour of leaf harvest and are hence potentially relevant for field-deployable applications.

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

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.

RNAi tools for controlling viroid diseases.

Viroids are small (250-400 nucleotides), single-stranded, circular RNAs without protein-coding capacity that infect a large number of ornamental and crop plant species, causing high economic losses worldwide. Strategies to control viroid diseases have included the use of naturally resistant cultivars in breeding programs, the superinfection exclusion with mild strains, the expression of ribonucleases, sense or antisense (catalytic) RNAs and, more recently, RNA interference (RNAi)-based tools. Here, I review the different RNAi strategies used to control viroid infections in plants, with particular focus on highly specific artificial small RNA (art-sRNA)-based tools such as artificial microRNAs and synthetic trans-acting small interfering RNAs. The advantages and future perspectives of art-sRNA-based RNAi for controlling viroid diseases are discussed.

Fine-Tuning Plant Gene Expression with Synthetic Trans-Acting Small Interfering RNAs.

RNAi-based tools are widely used in gene function studies and for crop improvement. However, no effective methods for precisely controlling the degree of induced silencing have been reported until recently. Here we report a detailed protocol for designing and generating synthetic trans-acting small interfering RNA (syn-tasiRNA) constructs for fine-tuning gene expression in plants. Recently developed high-throughput AtTAS1c-D2-B/c-based vectors are used to clone and express syn-tasiRNAs that possess different efficacies depending on their precursor location and on their degree of base-pairing with the 5' end of target RNAs.

Intact RNA structurome reveals mRNA structure-mediated regulation of miRNA cleavage in vivo.

MicroRNA (miRNA)-mediated cleavage is involved in numerous essential cellular pathways. miRNAs recognize target RNAs via sequence complementarity. In addition to complementarity, in vitro and in silico studies have suggested that RNA structure may influence the accessibility of mRNAs to miRNA-induced silencing complexes (miRISCs), thereby affecting RNA silencing. However, the regulatory mechanism of mRNA structure in miRNA cleavage remains elusive. We investigated the role of in vivo RNA secondary structure in miRNA cleavage by developing the new CAP-STRUCTURE-seq method to capture the intact mRNA structurome in Arabidopsis thaliana. This approach revealed that miRNA target sites were not structurally accessible for miRISC binding prior to cleavage in vivo. Instead, we found that the unfolding of the target site structure plays a key role in miRISC activity in vivo. We found that the single-strandedness of the two nucleotides immediately downstream of the target site, named Target Adjacent nucleotide Motif, can promote miRNA cleavage but not miRNA binding, thus decoupling target site binding from cleavage. Our findings demonstrate that mRNA structure in vivo can modulate miRNA cleavage, providing evidence of mRNA structure-dependent regulation of biological processes.

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

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.

Artificial Small RNA-Based Silencing Tools for Antiviral Resistance in Plants.

Artificial small RNAs (art-sRNAs), such as artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs), are highly specific 21-nucleotide small RNAs designed to recognize and silence complementary target RNAs. Art-sRNAs are extensively used in gene function studies or for improving crops, particularly to protect plants against viruses. Typically, antiviral art-sRNAs are computationally designed to target one or multiple sites in viral RNAs with high specificity, and art-sRNA constructs are generated and introduced into plants that are subsequently challenged with the target virus(es). Numerous studies have reported the successful application of art-sRNAs to induce resistance against a large number of RNA and DNA viruses in model and crop species. However, the application of art-sRNAs as an antiviral tool has limitations, such as the difficulty to predict the efficacy of a particular art-sRNA or the emergence of virus variants with mutated target sites escaping to art-sRNA-mediated degradation. Here, we review the different classes, features, and uses of art-sRNA-based tools to induce antiviral resistance in plants. We also provide strategies for the rational design of antiviral art-sRNAs and discuss the latest advances in developing art-sRNA-based methodologies for enhanced resistance to plant viruses.

Multi-targeting of viral RNAs with synthetic trans-acting small interfering RNAs enhances plant antiviral resistance.

RNA interference (RNAi)-based tools are used in multiple organisms to induce antiviral resistance through the sequence-specific degradation of target RNAs by complementary small RNAs. In plants, highly specific antiviral RNAi-based tools include artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs). syn-tasiRNAs have emerged as a promising antiviral tool allowing for the multi-targeting of viral RNAs through the simultaneous expression of several syn-tasiRNAs from a single precursor. Here, we compared in tomato plants the effects of an amiRNA construct expressing a single amiRNA and a syn-tasiRNA construct expressing four different syn-tasiRNAs against Tomato spotted wilt virus (TSWV), an economically important pathogen affecting tomato crops worldwide. Most of the syn-tasiRNA lines were resistant to TSWV, whereas the majority of the amiRNA lines were susceptible and accumulated viral progenies with mutations in the amiRNA target site. Only the two amiRNA lines with higher amiRNA accumulation were resistant, whereas resistance in syn-tasiRNA lines was not exclusive of lines with high syn-tasiRNA accumulation. Collectively, these results suggest that syn-tasiRNAs induce enhanced antiviral resistance because of the combined silencing effect of each individual syn-tasiRNA, which minimizes the possibility that the virus simultaneously mutates all different target sites to fully escape each syn-tasiRNA.

Identification and Characterization of Stress-Responsive TAS3-Derived TasiRNAs in Melon.

Small interfering RNAs (siRNA) are key regulators of gene expression that play essential roles in diverse biological processes. Trans-acting siRNAs (tasiRNAs) are a class of plant-endogenous siRNAs that lead the cleavage of nonidentical transcripts. TasiRNAs are usually involved in fine-tuning development. However, increasing evidence supports that tasiRNAs may be involved in stress response. Melon is a crop of great economic importance extensively cultivated in semiarid regions frequently exposed to changing environmental conditions that limit its productivity. However, knowledge of the precise role of siRNAs in general, and of tasiRNAs in particular, in regulating the response to adverse environmental conditions is limited. Here, we provide the first comprehensive analysis of computationally inferred melon-tasiRNAs responsive to two biotic (viroid-infection) and abiotic (cold treatment) stress conditions. We identify two TAS3-loci encoding to length (TAS3-L) and short (TAS3-S) transcripts. The TAS candidates predicted from small RNA-sequencing data were characterized according to their chromosome localization and expression pattern in response to stress. The functional activity of cmTAS genes was validated by transcript quantification and degradome assays of the tasiRNA precursors and their predicted targets. Finally, the functionality of a representative cmTAS3-derived tasiRNA (TAS3-S) was confirmed by transient assays showing the cleavage of ARF target transcripts.

Design, Synthesis, and Functional Analysis of Highly Specific Artificial Small RNAs with Antiviral Activity in Plants.

Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are two classes of artificial small RNAs (sRNAs) that have been broadly used to confer antiviral resistance in plants. However, methods for designing, synthesizing and functionally analyzing antiviral artificial sRNAs have not been optimized for time and cost-effectiveness and high-throughput applicability since recently. Here we present a systematic methodology for the simple and fast-forward design, generation, and functional analysis of large numbers of artificial sRNA constructs engineered to induce high levels of antiviral resistance in plants. Artificial sRNA constructs are transiently expressed in Nicotiana benthamiana plants, which are subsequently inoculated with the virus of interest. The antiviral activity of each artificial sRNA construct is assessed by monitoring viral symptom appearance, and through molecular analysis of virus accumulation in plant tissues. This approach is aimed to easily identify artificial sRNAs with high antiviral activity that could be expressed in transgenic plants for highly durable antiviral resistance.

Design and High-Throughput Generation of Artificial Small RNA Constructs for Plants.

Artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are two classes of 21-nucleotide artificial small RNAs (sRNAs) designed to selectively silence transcripts in plants with high efficacy and specificity. Despite their extensive use during the last decade, methods for designing and generating artificial sRNA constructs have not been optimized for time- and cost-effectiveness and high-throughput applicability since recently. In this chapter, I detail the protocols for both the rationale design and high-throughput generation of plant artificial sRNA constructs using the P-SAMS ("Plant Small RNA Maker Suite") web tool and a new generation of BsaI/ccdB (B/c) vectors optimized for one-step cloning of artificial sRNA inserts. These protocols allow for the efficient generation of large number of amiRNA and syn-tasiRNA constructs for potent, selective, and specific gene silencing in plants.

Fast-Forward Identification of Highly Effective Artificial Small RNAs Against Different Tomato spotted wilt virus Isolates.

Artificial small RNAs (sRNAs), including artificial microRNAs (amiRNAs) and synthetic trans-acting small interfering RNAs (syn-tasiRNAs), are used to silence viral RNAs and confer antiviral resistance in plants. Here, the combined use of recent high-throughput methods for generating artificial sRNA constructs and the Tomato spotted wilt virus (TSWV)-Nicotiana benthamiana pathosystem allowed for the simple and rapid identification of amiRNAs with high anti-TSWV activity. A comparative analysis between the most effective amiRNA construct and a syn-tasiRNA construct including the four most effective amiRNA sequences showed that both were highly effective against two different TSWV isolates. These results highlight the usefulness of this high-throughput methodology for the fast-forward identification of artificial sRNAs with high antiviral activity prior to time-consuming generation of stably transformed plants.

Plant ARGONAUTEs: Features, Functions, and Unknowns.

ARGONAUTEs (AGOs) are the effector proteins in eukaryotic small RNA (sRNA)-based gene silencing pathways controlling gene expression and transposon activity. In plants, AGOs regulate key biological processes such as development, response to stress, genome structure and integrity, and pathogen defense. Canonical functions of plant AGO-sRNA complexes include the endonucleolytic cleavage or translational inhibition of target RNAs and the methylation of target DNAs. Here, I provide a brief update on the major features, molecular functions, and biological roles of plant AGOs. A special focus is given to the more recent discoveries related to emerging molecular or biological functions of plant AGOs, as well as to the major unknowns in the plant AGO field.

Immunoprecipitation and High-Throughput Sequencing of ARGONAUTE-Bound Target RNAs from Plants.

ARGONAUTE (AGO) proteins function in small RNA (sRNA)-based RNA silencing pathways to regulate gene expression and control invading nucleic acids. In posttranscriptional RNA silencing pathways, plant AGOs associate with sRNAs to interact with highly sequence-complementary target RNAs. Once the AGO-sRNA-target RNA ternary complex is formed, target RNA is typically repressed through AGO-mediated cleavage or through other cleavage-independent mechanisms. The universe of sRNAs associating with diverse plant AGOs has been determined though AGO immunoprecipitation (IP) and high-throughput sequencing of co-immunoprecipitated sRNAs. To better understand the biological functions of AGO-sRNA complexes, it is crucial to identify the repertoire of target RNAs they regulate. Here I present a detailed AGO-RNA IP followed by high-throughput sequencing (AGO RIP-Seq) methodology for the isolation of AGO ternary complexes from plant tissues and the high-throughput sequencing of AGO-bound target RNAs. In particular, the protocol describes the IP of slicer-deficient hemagglutinin (HA)-tagged AGO proteins expressed in plant tissues, the isolation of AGO-bound RNAs, and the generation of target RNA libraries for high-throughput sequencing.

A viral suppressor of RNA silencing inhibits ARGONAUTE 1 function by precluding target RNA binding to pre-assembled RISC.

In most eukaryotes, RNA silencing is an adaptive immune system regulating key biological processes including antiviral defense. To evade this response, viruses of plants, worms and insects have evolved viral suppressors of RNA silencing proteins (VSRs). Various VSRs, such as P1 from Sweet potato mild mottle virus (SPMMV), inhibit the activity of RNA-induced silencing complexes (RISCs) including an ARGONAUTE (AGO) protein loaded with a small RNA. However, the specific mechanisms explaining this class of inhibition are unknown. Here, we show that SPMMV P1 interacts with AGO1 and AGO2 from Arabidopsis thaliana, but solely interferes with AGO1 function. Moreover, a mutational analysis of a newly identified zinc finger domain in P1 revealed that this domain could represent an effector domain as it is required for P1 suppressor activity but not for AGO1 binding. Finally, a comparative analysis of the target RNA binding capacity of AGO1 in the presence of wild-type or suppressor-defective P1 forms revealed that P1 blocks target RNA binding to AGO1. Our results describe the negative regulation of RISC, the small RNA containing molecular machine.