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Crocins are glucosylated apocarotenoids present in flowers and fruits of a few plant species, including saffron, gardenia, and Buddleja. The biosynthesis of crocins in these plants has been unraveled, and the enzymes engineered for the production of crocins in heterologous systems. Mullein (Verbascum sp.) has been identified as a new source of crocins and picrocrocin. In this work, we have identified eight enzymes involved in the cleavage of carotenoids in two Verbascum species, V. giganteum and V. sinuatum. Four of them were homologous to the previously identified BdCCD4.1 and BdCCD4.3 from Buddleja, involved in the biosynthesis of crocins. These enzymes were analyzed for apocarotenogenic activity in bacteria and Nicotiana benthamiana plants using a virus-driven system. Metabolic analyses of bacterial extracts and N. benthamiana leaves showed the efficient activity of these enzymes to produce crocins using β-carotene and zeaxanthin as substrates. Accumulations of 0.17% of crocins in N. benthamiana dry leaves were reached in only 2 weeks using a recombinant virus expressing VgCCD4.1, similar to the amounts previously produced using the canonical saffron CsCCD2L. The identification of these enzymes, which display a particularly broad substrate spectrum, opens new avenues for apocarotenoid biotechnological production., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Viral nanoparticles (VNPs) are a new class of virus-based formulations that can be used as building blocks to implement a variety of functions of potential interest in biotechnology and nanomedicine. Viral coat proteins (CP) that exhibit self-assembly properties are particularly appropriate for displaying antigens and antibodies, by generating multivalent VNPs with therapeutic and diagnostic potential. Here, we developed genetically encoded multivalent VNPs derived from two filamentous plant viruses, potato virus X (PVX) and tobacco etch virus (TEV), which were efficiently and inexpensively produced in the biofactory Nicotiana benthamiana plant. PVX and TEV-derived VNPs were decorated with two different nanobodies recognizing two different regions of the receptor-binding domain (RBD) of the SARS-CoV-2 Spike protein. The addition of different picornavirus 2A ribosomal skipping peptides between the nanobody and the CP allowed for modulating the degree of VNP decoration. Nanobody-decorated VNPs purified from N. benthamiana tissues successfully recognized the RBD antigen in enzyme-linked immunosorbent assays and showed efficient neutralization activity against pseudoviruses carrying the Spike protein. Interestingly, multivalent PVX and TEV-derived VNPs exhibited a neutralizing activity approximately one order of magnitude higher than the corresponding nanobody in a dimeric format. These properties, combined with the ability to produce VNP cocktails in the same N. benthamiana plant based on synergistic infection of the parent PVX and TEV, make these green nanomaterials an attractive alternative to standard antibodies for multiple applications in diagnosis and therapeutics., (© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Background: The Mediterranean fruit fly (medfly), Ceratitis capitata Wiedemann, is a major pest affecting fruit and vegetable production worldwide, whose control is mainly based on insecticides. Double-stranded RNA (dsRNA) able to down-regulate endogenous genes, thus affecting essential vital functions via RNA interference (RNAi) in pests and pathogens, is envisioned as a more specific and environmentally-friendly alternative to traditional insecticides. However, this strategy has not been explored in medfly yet., Results: Here, we screened seven candidate target genes by injecting in adult medflies gene-specific dsRNA hairpins transcribed in vitro. Several genes were significantly down-regulated, resulting in increased insect mortality compared to flies treated with a control dsRNA targeting the green fluorescent protein (GFP) complementary DNA (cDNA). Three of the dsRNAs, homologous to the beta subunit of adenosine triphosphate (ATP) synthase (ATPsynbeta), a vacuolar ATPase (V-ATPase), and the ribosomal protein S13 (RPS13), were able to halve the probability of survival in only 48 h after injection. We then produced new versions of these three dsRNAs and that of the GFP control as circular molecules in Escherichia coli using a two-self-splicing-intron-based expression system and tested them as orally-delivered insecticidal compounds against medfly adults. We observed a significant down-regulation of V-ATPase and RPS13 messenger RNAs (mRNAs) (approximately 30% and 90%, respectively) compared with the control medflies after 3 days of treatment. No significant mortality was recorded in medflies, but egg laying and hatching reduction was achieved by silencing V-ATPase and RPS13., Conclusion: In sum, we report the potential of dsRNA molecules as oral insecticide in medfly. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Insect pests rank among the major limiting factors in agricultural production worldwide. In addition to direct effect on crops, some phytophagous insects are efficient vectors for plant disease transmission. Large amounts of conventional insecticides are required to secure food production worldwide, with a high impact on the economy and environment, particularly when beneficial insects are also affected by chemicals that frequently lack the desired specificity. RNA interference (RNAi) is a natural mechanism gene expression regulation and protection against exogenous and endogenous genetic elements present in most eukaryotes, including insects. Molecules of double-stranded RNA (dsRNA) or highly structured RNA are the substrates of cellular enzymes to produce several types of small RNAs (sRNAs), which play a crucial role in targeting sequences for transcriptional or post-transcriptional gene silencing. The relatively simple rules that underlie RNAi regulation, mainly based in Watson-Crick complementarity, have facilitated biotechnological applications based on these cellular mechanisms. This includes the promise of using engineered dsRNA molecules, either endogenously produced in crop plants or exogenously synthesized and applied onto crops, as a new generation of highly specific, sustainable, and environmentally friendly insecticides. Fueled on this expectation, this article reviews current knowledge about the RNAi pathways in insects, and some other applied questions such as production and delivery of recombinant RNA, which are critical to establish RNAi as a reliable technology for insect control in crop plants.

Virus-induced genome editing (VIGE) leverages viral vectors to deliver CRISPR-Cas components into plants for robust and flexible trait engineering. We describe here a VIGE approach applying an RNA viral vector based on potato virus X (PVX) for genome editing of tomato, a mayor horticultural crop. Viral delivery of single-guide RNA into Cas9-expressing lines resulted in efficient somatic editing with indel frequencies up to 58%. By proof-of-concept VIGE of PHYTOENE DESATURASE ( PDS ) and plant regeneration from edited somatic tissue, we recovered loss-of-function pds mutant progeny displaying an albino phenotype. VIGE of STAYGREEN 1 ( SGR1 ), a gene involved in fruit color variation, generated sgr1 mutant lines with recolored red-brown fruits and high chlorophyll levels. The obtained editing events were heritable, overall confirming the successful breeding of fruit color. Altogether, our VIGE approach offers great potential for accelerated functional genomics of tomato variation, as well as for precision breeding of novel tomato traits., Competing Interests: The authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nanjing Agricultural University.)

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., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Recurrent disease outbreaks caused by different viruses, including the novel respiratory virus SARS-CoV-2, are challenging our society at a global scale; so versatile virus detection methods would enable a calculated and faster response. Here, we present a novel nucleic acid detection strategy based on CRISPR-Cas9, whose mode of action relies on strand displacement rather than on collateral catalysis, using the Streptococcus pyogenes Cas9 nuclease. Given a preamplification process, a suitable molecular beacon interacts with the ternary CRISPR complex upon targeting to produce a fluorescent signal. We show that SARS-CoV-2 DNA amplicons generated from patient samples can be detected with CRISPR-Cas9. We also show that CRISPR-Cas9 allows the simultaneous detection of different DNA amplicons with the same nuclease, either to detect different SARS-CoV-2 regions or different respiratory viruses. Furthermore, we demonstrate that engineered DNA logic circuits can process different SARS-CoV-2 signals detected by the CRISPR complexes. Collectively, this CRISPR-Cas9 R-loop usage for the molecular beacon opening (COLUMBO) platform allows a multiplexed detection in a single tube, complements the existing CRISPR-based methods, and displays diagnostic and biocomputing potential.

Crocins are glycosylated apocarotenoids with strong coloring power and anti-oxidant, anticancer, and neuro-protective properties. We previously dissected the saffron crocin biosynthesis pathway, and demonstrated that the CsCCD2 enzyme, catalyzing the carotenoid cleavage step, shows a strong preference for the xanthophyll zeaxanthin in vitro and in bacterio. In order to investigate substrate specificity in planta and to establish a plant-based bio-factory system for crocin production, we compared wild-type Nicotiana benthamiana plants, accumulating various xanthophylls together with α- and β-carotene, with genome-edited lines, in which all the xanthophylls normally accumulated in leaves were replaced by a single xanthophyll, zeaxanthin. These plants were used as chassis for the production in leaves of saffron apocarotenoids (crocins, picrocrocin) using two transient expression methods to overexpress CsCCD2 : agroinfiltration and inoculation with a viral vector derived from tobacco etch virus (TEV). The results indicated the superior performance of the zeaxanthin-accumulating line and of the use of the viral vector to express CsCCD2 . The results also suggested a relaxed substrate specificity of CsCCD2 in planta, cleaving additional carotenoid substrates.

The recent emergence of tools based on the clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins have revolutionized targeted genome editing, thus holding great promise to both basic plant science and precision crop breeding. Conventional approaches for the delivery of editing components rely on transformation technologies or transient delivery to protoplasts, both of which are time-consuming, laborious, and can raise legal concerns. Alternatively, plant RNA viruses can be used as transient delivery vectors of CRISPR-Cas reaction components, following the so-called virus-induced genome editing (VIGE). During the last years, researchers have been able to engineer viral vectors for the delivery of CRISPR guide RNAs and Cas nucleases. Considering that each viral vector is limited to its molecular biology properties and a specific host range, here we review recent advances for improving the VIGE toolbox with a special focus on strategies to achieve tissue-culture-free editing in plants. We also explore the utility of CRISPR-Cas technology to enhance biotic resistance with a special focus on plant virus diseases. This can be achieved by either targeting the viral genome or modifying essential host susceptibility genes that mediate in the infection process. Finally, we discuss the challenges and potential that VIGE holds in future breeding technologies., (© 2022 The Authors. The Plant Genome published by Wiley Periodicals LLC on behalf of Crop Science Society of America.)

Viral vectors hold enormous potential for genome editing in plants as transient delivery vehicles of CRISPR-Cas components. Here, we describe a protocol to assemble plant viral vectors for single-guide RNA (sgRNA) delivery. The obtained viral constructs are based on compact T-DNA binary vectors of the pLX series and are delivered into Cas9-expressing plants through agroinoculation. This approach allows rapidly assessing sgRNA design for plant genome targeting, as well as the recovery of progeny with heritable mutations at targeted loci. For complete details on the use and execution of this protocol, please refer to Uranga et al. (2021) 1 and Aragonés et al. (2022). 2 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Viroids are a unique type of infectious agent, exclusively composed of a relatively small (246-430 nt), highly base-paired, circular, non-coding RNA. Despite the small size and non-coding nature, the more-than-thirty currently known viroid species infectious of higher plants are able to autonomously replicate and move systemically through the host, thereby inducing disease in some plants. After recalling viroid discovery back in the late 60s and early 70s of last century and discussing current hypotheses about their evolutionary origin, this article reviews our current knowledge about these peculiar infectious agents. We describe the highly base-paired viroid molecules that fold in rod-like or branched structures and viroid taxonomic classification in two families, Pospiviroidae and Avsunviroidae , likely gathering nuclear and chloroplastic viroids, respectively. We review current knowledge about viroid replication through RNA-to-RNA rolling-circle mechanisms in which host factors, notably RNA transporters, RNA polymerases, RNases, and RNA ligases, are involved. Systemic movement through the infected plant, plant-to-plant transmission and host range are also discussed. Finally, we focus on the mechanisms of viroid pathogenesis, in which RNA silencing has acquired remarkable importance, and also for the initiation of potential biotechnological applications of viroid molecules.

A gene construct encoding a xylanase, which is active in extreme conditions of temperature and alkaline pH (90 °C, pH 10.5), has been transitorily expressed with high efficiency in Nicotiana benthamiana using a viral vector. The enzyme, targeted to the apoplast, accumulates in large amounts in plant tissues in as little as 7 days after inoculation, without detrimental effects on plant growth. The properties of the protein produced by the plant, in terms of resistance to temperature, pH, and enzymatic activity, are equivalent to those observed when Escherichia coli is used as a host. Purification of the plant-produced recombinant xylanase is facilitated by exporting the protein to the apoplastic space. The production of this xylanase by N. benthamiana, which avoids the hindrances derived from the use of E. coli, namely, intracellular production requiring subsequent purification, represents an important step for potential applications in the food industry in which more sustainable and green products are continuously demanded. As an example, the use of the enzyme producing prebiotic xylooligosdaccharides from xylan is here reported., (© 2022. The Author(s).)

Programmable transcriptional regulators based on CRISPR architecture are promising tools for the induction of plant gene expression. In plants, CRISPR gene activation is effective with respect to modulating development processes, such as the flowering time or customizing biochemical composition. The most widely used method for delivering CRISPR components into the plant is Agrobacterium tumefaciens-mediated genetic transformation, either transient or stable. However, as a result of their versatility and their ability to move, virus-derived systems have emerged as an interesting alternative for supplying the CRISPR components to the plant, in particular guide RNA (gRNA), which represents the variable component in CRISPR strategies. In the present study, we describe a Potato virus X-derived vector that, upon agroinfection in Nicotiana benthamiana, serves as a vehicle for delivery of gRNAs, producing highly specific virus-induced gene activation. The system works in combination with a N. benthamiana transgenic line carrying the remaining complementary CRISPR gene activation components, specifically the dCasEV2.1 cassette, which has been shown previously to mediate strong programmable transcriptional activation in plants. Using an easily scalable, non-invasive spraying method, we show that gRNA-mediated activation programs move locally and systemically, generating a strong activation response in different target genes. Furthermore, by activating three different endogenous MYB transcription factors, we demonstrate that this Potato virus X-based virus-induced gene reprogramming strategy results in program-specific metabolic fingerprints in N. benthamiana leaves characterized by distinctive phenylpropanoid-enriched metabolite profiles., (© 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

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.

Viral vectors provide a quick and effective way to express exogenous sequences in eukaryotic cells and to engineer eukaryotic genomes through the delivery of CRISPR/Cas components. Here, we present JoinTRV, an improved vector system based on tobacco rattle virus (TRV) that simplifies gene silencing and genome editing logistics. Our system consists of two mini T-DNA vectors from which TRV RNA1 (pLX-TRV1) and an engineered version of TRV RNA2 (pLX-TRV2) are expressed. The two vectors have compatible origins that allow their cotransformation and maintenance into a single Agrobacterium cell, as well as their simultaneous delivery to plants by a one-Agrobacterium/two-vector approach. The JoinTRV vectors are substantially smaller than those of any known TRV vector system, and pLX-TRV2 can be easily customized to express desired sequences by one-step digestion-ligation and homology-based cloning. The system was successfully used in Nicotiana benthamiana for launching TRV infection, for recombinant protein production, as well as for robust virus-induced gene silencing (VIGS) of endogenous transcripts using bacterial suspensions at low optical densities. JoinTRV-mediated delivery of single-guide RNAs in a Cas9 transgenic host allowed somatic cell editing efficiencies of ≈90%; editing events were heritable and >50% of the progeny seedlings showed mutations at the targeted loci., (© 2021 The Authors. Biotechnology Journal published by Wiley-VCH GmbH.)

Potyviridae, the largest family of known RNA viruses (realm Riboviria), belongs to the picorna-like supergroup and has important agricultural and ecological impacts. Potyvirid genomes are translated into polyproteins, which are in turn hydrolyzed to release mature products. Recent sequencing efforts revealed an unprecedented number of potyvirids with a rich variability in gene content and genomic layouts. Here, we review the heterogeneity of non-core modules that expand the structural and functional diversity of the potyvirid proteomes. We provide a family-wide classification of P1 proteinases into the functional Types A and B, and discuss pretty interesting sweet potato potyviral ORF (PISPO), putative zinc fingers, and alkylation B (AlkB)-non-core modules found within P1 cistrons. The atypical inosine triphosphate pyrophosphatase (ITPase/HAM1), as well as the pseudo tobacco mosaic virus-like coat protein (TMV-like CP) are discussed alongside homologs of unrelated virus taxa. Family-wide abundance of the multitasking helper component proteinase (HC-pro) is revised. Functional connections between non-core modules are highlighted to support host niche adaptation and immune evasion as main drivers of the Potyviridae evolutionary radiation. Potential biotechnological and synthetic biology applications of potyvirid leader proteinases and non-core modules are finally explored., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

Background: Carotenoids are health-promoting metabolites in livestock and human diets. Some important crops have been genetically modified to increase their content. Although the usefulness of transgenic plants to alleviate nutritional deficiencies is obvious, their social acceptance has been controversial., Results: Here, we demonstrate an alternative biotechnological strategy for carotenoid fortification of edible fruits in which no transgenic DNA is involved. A viral RNA vector derived from zucchini yellow mosaic virus (ZYMV) was modified to express a bacterial phytoene synthase (crtB), and inoculated to zucchini (Cucurbita pepo L.) leaves nurturing pollinated flowers. After the viral vector moved to the developing fruit and expressed crtB, the rind and flesh of the fruits developed yellow-orange rather than green color. Metabolite analyses showed a substantial enrichment in health-promoting carotenoids, such as α- and β-carotene (provitamin A), lutein and phytoene, in both rind and flesh., Conclusion: Although this strategy is perhaps not free from controversy due to the use of genetically modified viral RNA, our work does demonstrate the possibility of metabolically fortifying edible fruits using an approach in which no transgenes are involved., (© 2022 Wiley-VCH GmbH.)

Enrichment of foodstuffs with health-promoting metabolites such as carotenoids is a powerful tool to fight against unhealthy eating habits. Dietary carotenoids are vitamin A precursors and reduce risk of several chronical diseases. Additionally, carotenoids and their cleavage products (apocarotenoids) are used as natural pigments and flavors by the agrofood industry. In the last few years, major advances have been made in our understanding of how plants make and store carotenoids in their natural compartments, the plastids. In part, this knowledge has been acquired by using transient expression systems, notably agroinfiltration and viral vectors. These techniques allow profound changes in the carotenoid profile of plant tissues at the desired developmental stage, hence preventing interference with normal plant growth and development. Here we review how transient expression approaches have contributed to learn about the structure and regulation of plant carotenoid biosynthesis and to rewire carotenoid metabolism and storage for efficient biofortification of plant tissues., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Viral nanoparticles (VNPs) have recently attracted attention for their use as building blocks for novel materials to support a range of functions of potential interest in nanotechnology and medicine. Viral capsids are ideal for presenting small epitopes by inserting them at an appropriate site on the selected coat protein (CP). VNPs presenting antibodies on their surfaces are considered highly promising tools for therapeutic and diagnostic purposes. Due to their size, nanobodies are an interesting alternative to classic antibodies for surface presentation. Nanobodies are the variable domains of heavy-chain (VHH) antibodies from animals belonging to the family Camelidae, which have several properties that make them attractive therapeutic molecules, such as their small size, simple structure, and high affinity and specificity. In this work, we have produced genetically encoded VNPs derived from two different potyviruses-the largest group of RNA viruses that infect plants-decorated with nanobodies. We have created a VNP derived from zucchini yellow mosaic virus (ZYMV) decorated with a nanobody against the green fluorescent protein (GFP) in zucchini ( Cucurbita pepo ) plants. As reported for other viruses, the expression of ZYMV-derived VNPs decorated with this nanobody was only made possible by including a picornavirus 2A splicing peptide between the fused proteins, which resulted in a mixed population of unmodified and decorated CPs. We have also produced tobacco etch virus (TEV)-derived VNPs in Nicotiana benthamiana plants decorated with the same nanobody against GFP. Strikingly, in this case, VNPs could be assembled by direct fusion of the nanobody to the viral CP with no 2A splicing involved, likely resulting in fully decorated VNPs. For both expression systems, correct assembly and purification of the recombinant VNPs was confirmed by transmission electron microscope; the functionality of the CP-fused nanobody was assessed by western blot and binding assays. In sum, here we report the production of genetically encoded plant-derived VNPs decorated with a nanobody. This system may be an attractive alternative for the sustainable production in plants of nanobody-containing nanomaterials for diagnostic and therapeutic purposes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer HP declared a past collaboration with one of the authors MM to the handling editor., (Copyright © 2022 Martí, Merwaiss, Butković and Daròs.)

CRISPR/Cas has revolutionized genome engineering in plants. However, the use of anti-CRISPR proteins as tools to prevent CRISPR/Cas-mediated gene editing and gene activation in plants has not been explored yet. This study describes the characterization of two anti-CRISPR proteins, AcrIIA4 and AcrVA1, in Nicotiana benthamiana. Our results demonstrate that AcrIIA4 prevents site-directed mutagenesis in leaves when transiently co-expressed with CRISPR/Cas9. In a similar way, AcrVA1 is able to prevent CRISPR/Cas12a-mediated gene editing. Moreover, using a N. benthamiana line constitutively expressing Cas9, we show that the viral delivery of AcrIIA4 using Tobacco etch virus is able to completely abolish the high editing levels obtained when the guide RNA is delivered with a virus, in this case Potato virus X. We also show that AcrIIA4 and AcrVA1 repress CRISPR/dCas-based transcriptional activation of reporter genes. In the case of AcrIIA4, this repression occurs in a highly efficient, dose-dependent manner. Furthermore, the fusion of an auxin degron to AcrIIA4 results in auxin-regulated activation of a downstream reporter gene. The strong anti-Cas activity of AcrIIA4 and AcrVA1 reported here opens new possibilities for customized control of gene editing and gene expression in plants., (© 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

The circular and linear forms of viroid RNAs can be separated by two-dimensional polyacrylamide gel electrophoresis (PAGE) based on the selective delay in mobility that circular RNAs experience under denaturing conditions. First PAGE separates RNA preparations from viroid-infected plants, and the whole lane from this first gel is next perpendicularly loaded on top of a second gel. Separation continues under new conditions that differ in the degree of denaturation from the first. The result is a two-dimensional separation of the RNAs in which circular and linear molecules are distributed in two parallel diagonals., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

Similar to viruses, viroids can also be engineered and transformed into useful biotechnological tools. We describe here a viroid-based system to produce large amounts of recombinant RNA in Escherichia coli. A precursor of eggplant latent viroid (ELVd), with the RNA of interest inserted between positions U245 and U246, is co-expressed in E. coli along the chloroplastic isoform of the eggplant tRNA ligase, the enzyme that mediates the circularization of this viroid in the infected plants. In the bacterial cells, the chimeric ELVd-RNA-of-interest precursor self-cleaves through the embedded hammerhead ribozymes, and the monomer is recognized and circularized by the co-expressed tRNA ligase. The resulting circular RNA, likely bound to the tRNA ligase, accumulates to a high concentration in the bacterial cells., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2022. Springer Science+Business Media, LLC, part of Springer Nature.)

RNA interference (RNAi) is a natural mechanism for protecting against harmful genetic elements and regulating gene expression, which can be artificially triggered by the delivery of homologous double-stranded RNA (dsRNA). This mechanism can be exploited as a highly specific and environmentally friendly pest control strategy. To this aim, systems for producing large amounts of recombinant dsRNA are necessary. We describe a system to efficiently produce large amounts of circular dsRNA in Escherichia coli and demonstrate the efficient insecticidal activity of these molecules against Western corn rootworm (WCR, Diabrotica virgifera virgifera LeConte), a highly damaging pest of corn crops. In our system, the two strands of the dsRNA are expressed in E. coli embedded within the very stable scaffold of Eggplant latent viroid (ELVd), a small circular non-coding RNA. Stability in E. coli of the corresponding plasmids with long inverted repeats was achieved by using a cDNA coding for a group-I autocatalytic intron from Tetrahymena thermophila as a spacer. RNA circularization and large-scale accumulation in E. coli cells was facilitated by co-expression of eggplant tRNA ligase, the enzyme that ligates ELVd during replication in the host plant. The inserted intron efficiently self-spliced from the RNA product during transcription. Circular RNAs containing a dsRNA moiety homologous to smooth septate junction 1 ( DvSSJ1 ) gene exhibited excellent insecticide activity against WCR larvae. Finally, we show that the viroid scaffold can be separated from the final circular dsRNA product using a second T. thermophila self-splicing intron in a permuted form.

The use of viral vectors that can replicate and move systemically through the host plant to deliver bacterial CRISPR components enables genome editing at the whole-plant level and avoids the requirement for labor-intensive stable transformation. However, this approach usually relies on previously transformed plants that stably express a CRISPR-Cas nuclease. Here, we describe successful DNA-free genome editing of Nicotiana benthamiana using two compatible RNA virus vectors derived from tobacco etch virus (TEV; genus Potyvirus ) and potato virus X (PVX; genus Potexvirus ), which replicate in the same cells. The TEV and PVX vectors respectively express a Cas12a nuclease and the corresponding guide RNA. This novel two-virus vector system improves the toolbox for transformation-free virus-induced genome editing in plants and will advance efforts to breed more nutritious, resistant, and productive crops.

As genomic architectures become more complex, they begin to accumulate degenerate and redundant elements. However, analyses of the molecular mechanisms underlying these genetic architecture features remain scarce, especially in compact but sufficiently complex genomes. In the present study, we followed a proteomic approach together with a computational network analysis to reveal molecular signatures of protein function degeneracy from a plant virus (as virus-host protein-protein interactions). We employed affinity purification coupled to mass spectrometry to detect several host factors interacting with two proteins of Citrus tristeza virus (p20 and p25) that are known to function as RNA silencing suppressors, using an experimental system of transient expression in a model plant. The study was expanded by considering two different isolates of the virus, and some key interactions were confirmed by bimolecular fluorescence complementation assays. We found that p20 and p25 target a common set of plant proteins including chloroplastic proteins and translation factors. Moreover, we noted that even specific targets of each viral protein overlap in function. Notably, we identified argonaute proteins (key players in RNA silencing) as reliable targets of p20. Furthermore, we found that these viral proteins preferentially do not target hubs in the host protein interactome, but elements that can transfer information by bridging different parts of the interactome. Overall, our results demonstrate that two distinct proteins encoded in the same viral genome that overlap in function also overlap in their interactions with the cell proteome, thereby highlighting an overlooked connection from a degenerate viral system., Competing Interests: The authors have declared that no competing interests exist.

DNA nanotechnology, and DNA computing in particular, has grown extensively over the past decade to end with a variety of functional stable structures and dynamic circuits. However, the use as designer elements of regular DNA pieces, perfectly complementary double strands, has remained elusive. Here, we report the exploitation of CRISPR-Cas systems to engineer logic circuits based on isothermal strand displacement that perform with toehold-free double-stranded DNA. We designed and implemented molecular converters for signal detection and amplification, showing good interoperability between enzymatic and nonenzymatic processes. Overall, these results contribute to enlarge the repertoire of substrates and reactions (hardware) for DNA computing.

Systems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR-associated proteins (Cas) have revolutionized genome editing in many organisms, including plants. Most CRISPR-Cas strategies in plants rely on genetic transformation using Agrobacterium tumefaciens to supply the gene editing reagents, such as Cas nucleases or the synthetic guide RNA (sgRNA). While Cas nucleases are constant elements in editing approaches, sgRNAs are target-specific and a screening process is usually required to identify those most effective. Plant virus-derived vectors are an alternative for the fast and efficient delivery of sgRNAs into adult plants, due to the virus capacity for genome amplification and systemic movement, a strategy known as virus-induced genome editing. We engineered Potato virus X (PVX) to build a vector that easily expresses multiple sgRNAs in adult solanaceous plants. Using the PVX-based vector, Nicotiana benthamiana genes were efficiently targeted, producing nearly 80% indels in a transformed line that constitutively expresses Streptococcus pyogenes Cas9. Interestingly, results showed that the PVX vector allows expression of arrays of unspaced sgRNAs, achieving highly efficient multiplex editing in a few days in adult plant tissues. Moreover, virus-free edited progeny can be obtained from plants regenerated from infected tissues or infected plant seeds, which exhibit a high rate of heritable biallelic mutations. In conclusion, this new PVX vector allows easy, fast and efficient expression of sgRNA arrays for multiplex CRISPR-Cas genome editing and will be a useful tool for functional gene analysis and precision breeding across diverse plant species, particularly in Solanaceae crops., (© 2021 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Viroids are relatively small, noncoding, plant circular RNAs. In contrast to other plant circular RNAs of endogenous origin, viroids are infectious agents able to replicate autonomously in the appropriate host. Because of their highly base-paired structures, they can be purified from infected tissue extracts using nonionic CF11 chromatography. Depending on the host plant species, viroid RNA preparation may also require polysaccharide removal by an extraction with 2-methoxyethanol followed by precipitation with cetyltrimethylammonium bromide. Electrophoretic analyses of this kind of preparations frequently show differential bands corresponding to the viroid circular molecules, which are absent in those from healthy plants. These RNA preparations can also be used for viroid transmission to new plants by mechanical inoculation.

Viroids are small circular, noncoding, highly base-paired RNAs able to infect higher plants. Recently, it has been shown that viroids can be used as very stable scaffolds to produce recombinant RNA in Escherichia coli. Coexpression of an RNA precursor consisting of a viroid monomer, in which the RNA of interest is inserted, flanked by domains of the viroid hammerhead ribozyme, along with a host plant tRNA ligase, the enzyme that catalyzes viroid circularization in infected plants, allows for accumulation of large amounts of the chimeric viroid-RNA of interest in E. coli. Since viroids do not replicate in E. coli, high accumulation most probably results from viroid scaffold stability, resistance to exonucleases due to circularity, and accumulation as a ribonucleoprotein complex with tRNA ligase. Purification of the recombinant RNA from total E. coli RNA is also facilitated by the circular structure of the product.

Crocins and picrocrocin are glycosylated apocarotenoids responsible, respectively, for the color and the unique taste of the saffron spice, known as red gold due to its high price. Several studies have also shown the health-promoting properties of these compounds. However, their high costs hamper the wide use of these metabolites in the pharmaceutical sector. We have developed a virus-driven system to produce remarkable amounts of crocins and picrocrocin in adult Nicotiana benthamiana plants in only two weeks. The system consists of viral clones derived from tobacco etch potyvirus that express specific carotenoid cleavage dioxygenase (CCD) enzymes from Crocus sativus and Buddleja davidii. Metabolic analyses of infected tissues demonstrated that the sole virus-driven expression of C. sativus CsCCD2L or B. davidii BdCCD4.1 resulted in the production of crocins, picrocrocin and safranal. Using the recombinant virus that expressed CsCCD2L, accumulations of 0.2% of crocins and 0.8% of picrocrocin in leaf dry weight were reached in only two weeks. In an attempt to improve apocarotenoid content in N. benthamiana, co-expression of CsCCD2L with other carotenogenic enzymes, such as Pantoea ananatis phytoene synthase (PaCrtB) and saffron β-carotene hydroxylase 2 (BCH2), was performed using the same viral system. This combinatorial approach led to an additional crocin increase up to 0.35% in leaves in which CsCCD2L and PaCrtB were co-expressed. Considering that saffron apocarotenoids are costly harvested from flower stigma once a year, and that Buddleja spp. flowers accumulate lower amounts, this system may be an attractive alternative for the sustainable production of these appreciated metabolites., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Plastids, the defining organelles of plant cells, undergo physiological and morphological changes to fulfill distinct biological functions. In particular, the differentiation of chloroplasts into chromoplasts results in an enhanced storage capacity for carotenoids with industrial and nutritional value such as beta-carotene (provitamin A). Here, we show that synthetically inducing a burst in the production of phytoene, the first committed intermediate of the carotenoid pathway, elicits an artificial chloroplast-to-chromoplast differentiation in leaves. Phytoene overproduction initially interferes with photosynthesis, acting as a metabolic threshold switch mechanism that weakens chloroplast identity. In a second stage, phytoene conversion into downstream carotenoids is required for the differentiation of chromoplasts, a process that involves a concurrent reprogramming of nuclear gene expression and plastid morphology for improved carotenoid storage. We hence demonstrate that loss of photosynthetic competence and enhanced production of carotenoids are not just consequences but requirements for chloroplasts to differentiate into chromoplasts., Competing Interests: The authors declare no competing interest.

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., (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Sustainable use of nanotechnology in crop protection requires an understanding of the plant's life cycle, potential toxicological impacts of nanomaterials and their mechanism of action against the target pathogens. Herein, we show some properties of a candidate antifungal nanocomposite made from copper oxide (CuO; otherwise an essential soil nutrient) nanoparticles (NPs), with definite size and shape, decorating the surface of reduced graphene oxide (rGO) nanosheets. The successful preparation of the rGO-CuO NPs was confirmed by spectroscopic and microscopic analyses, and its antifungal activity against wild strains of Fusarium oxysporum affecting tomato and pepper plants was successfully confirmed. A comparative analysis in vitro indicated that this nanocomposite had higher antifungal activity at only 1 mg/L than the conventional fungicide Kocide 2000 at 2.5 g/L. Further investigation suggested that rGO-CuO NPs creates pits and pores on the fungal cell membranes inducing cell death. In planta results indicated that only 1 mg/L from the nanocomposite is required to reduce Fusarium wilt and root rot diseases severity below 5% for tomato and pepper plants without any phytotoxicity for about 70 days. Comparatively, 2.5 g/L of Kocide 2000 are required to achieve about 30% disease reduction in both plants. The present study contributes to the concept of agro-nanotechnology, showing the properties of a novel ecofriendly and economic nanopesticide for sustainable plant protection.

Main Conclusion: MiMYB1 and MibHLH2 play key roles in anthocyanin biosynthesis in Matthiola incana flowers. We established a transient expression system using Turnip mosaic virus vector in M. incana. Garden stock (Matthiola incana (L.) R. Br.) is a popular flowering plant observed from winter to spring in Japan. Here we observed that anthocyanin accumulation in 'Vintage Lavender' increased with flower development, whereas flavonol accumulation remained constant throughout flower development. We obtained five transcription factor genes, MiMYB1, MibHLH1, MibHLH2, MiWDR1, and MiWDR2, from M. incana floral cDNA contigs. Yeast two-hybrid analyses revealed that MiMYB1 interacted with MibHLH1, MibHLH2, and MiWDR1, but MiWDR2 did not interact with any transcription factor. Expression levels of MiMYB1 and MibHLH2 increased in petals during floral bud development. Their expression profiles correlated well with the temporal profiles of MiF3'H, MiDFR, MiANS, and Mi3GT transcripts and anthocyanin accumulation profile. On the other hand, MibHLH1 was expressed weakly in all organs of 'Vintage Lavender'. However, high expression levels of MibHLH1 were detected in petals of other cultivars with higher levels of anthocyanin accumulation than 'Vintage Lavender'. MiWDR1 and MiWDR2 maintained constant expression levels in petals during flower development and vegetative organs. Transient MiMYB1 expression in 1-month-old M. incana seedlings using a Turnip mosaic virus vector activated transcription of the endogenous anthocyanin biosynthetic genes MiF3'H, MiDFR, and MiANS and induced ectopic anthocyanin accumulation in leaves. Therefore, MiMYB1 possibly interacts with MibHLH2 and MiWDR1, and this trimeric protein complex activates the transcription of anthocyanin biosynthetic genes in M. incana flowers. Moreover, MibHLH1 acts as an enhancer of anthocyanin biosynthesis with the MiMYB1-MibHLH2-MiWDR1 complex. This study revealed the molecular mechanism involved in the regulation of anthocyanin accumulation levels in M. incana flowers.

Potyviruses are plus-strand RNA viruses that can be easily transformed into expression vectors to quickly express one carotenogenic enzyme or transcription factor, or more, in plant tissues. Unlike the technically challenging and time-consuming process of plant transformation, manipulation of a roughly 10,000 nt-long viral genome is rather straightforward via common molecular biology techniques. Here I describe how to insert the cDNAs of the proteins of interest into two particular positions of the cDNA of a Tobacco etch virus (TEV) mutant that lacks the viral NIb cistron and only infects the plants in which this protein is expressed. This deletion increases the space to harbor foreign sequences. The selection of the expression site must be made according to subcellular localization requirements. The recombinant virus is then inoculated into Nicotiana benthamiana plants by means of Agrobacterium tumefaciens. The expression of the viral genome entails the production of carotenogenic proteins in the plant tissues with a consequent effect on the plant carotenoid pathway.

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., (© 2019 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Fungi that infect plants, animals or humans pose a serious threat to human health and food security. Antifungal proteins (AFPs) secreted by filamentous fungi are promising biomolecules that could be used to develop new antifungal therapies in medicine and agriculture. They are small highly stable proteins with specific potent activity against fungal pathogens. However, their exploitation requires efficient, sustainable and safe production systems. Here, we report the development of an easy-to-use, open access viral vector based on Tobacco mosaic virus (TMV). This new system allows the fast and efficient assembly of the open reading frames of interest in small intermediate entry plasmids using the Gibson reaction. The manipulated TMV fragments are then transferred to the infectious clone by a second Gibson assembly reaction. Recombinant proteins are produced by agroinoculating plant leaves with the resulting infectious clones. Using this simple viral vector, we have efficiently produced two different AFPs in Nicotiana benthamiana leaves, namely the Aspergillus giganteus AFP and the Penicillium digitatum AfpB. We obtained high protein yields by targeting these bioactive small proteins to the apoplastic space of plant cells. However, when AFPs were targeted to intracellular compartments, we observed toxic effects in the host plants and undetectable levels of protein. We also demonstrate that this production system renders AFPs fully active against target pathogens, and that crude plant extracellular fluids containing the AfpB can protect tomato plants from Botrytis cinerea infection, thus supporting the idea that plants are suitable biofactories to bring these antifungal proteins to the market., (© 2018 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Recent metagenomic studies have provided an unprecedented wealth of data, which are revolutionizing our understanding of virus diversity. A redrawn landscape highlights viruses as active players in the phytobiome, and surveys have uncovered their positive roles in environmental stress tolerance of plants. Viral infectious clones are key tools for functional characterization of known and newly identified viruses. Knowledge of viruses and their components has been instrumental for the development of modern plant molecular biology and biotechnology. In this review, we provide extensive guidelines built on current synthetic biology advances that streamline infectious clone assembly, thus lessening a major technical constraint of plant virology. The focus is on generation of infectious clones in binary T-DNA vectors, which are delivered efficiently to plants by Agrobacterium. We then summarize recent applications of plant viruses and explore emerging trends in microbiology, bacterial and human virology that, once translated to plant virology, could lead to the development of virus-based gene therapies for ad hoc engineering of plant traits. The systematic characterization of plant virus roles in the phytobiome and next-generation virus-based tools will be indispensable landmarks in the synthetic biology roadmap to better crops., (© 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Composed of a few hundreds of nucleotides, viroids are infectious, circular, non-protein coding RNAs able to usurp plant cellular enzymes and molecular machineries to replicate and move in their hosts. Several secondary and tertiary RNA structural motifs have been implicated in the viroid infectious cycle, but whether modified nucleotides, such as 5C-methylcytosine (m 5 C), also play a role has not been deeply investigated so far. Here, the possible existence of m 5 C in both RNA polarity strands of potato spindle tuber viroid and avocado sunblotch viroid -which are representative members of the nucleus- and chloroplast-replicating viroids, respectively- has been assessed at single nucleotide level. We show that a standard bisulfite protocol efficiently used for identifying m 5 C in cellular RNAs may generate false positive results in the case of the highly structured viroid RNAs. Applying a bisulfite conversion protocol specifically adapted to RNAs with high secondary structure, no m 5 C was identified in both polarity strands of both viroids, indicating that this specific nucleotide modification does not likely play a role in viroid biology.

The stunting disease, incited by chrysanthemum stunt viroid (CSVd), has become a serious problem in chrysanthemum production areas worldwide. Here we identified 46 weed species from chrysanthemum fields in two producing regions of the State of São Paulo, Brazil. The mechanical inoculation of these weeds with a Brazilian CSVd isolate revealed that this viroid was able to infect 17 of these species, in addition to chrysanthemum, tomato and potato. Plants of Oxalis latifolia and chrysanthemum naturally infected with CSVd were found in chrysanthemum fields in Colombia, which is the first CSVd report in that country. Therefore, weeds have the potential to act as reservoirs of CSVd in the field. These results are the first reports of experimental CSVd infection in the following species: Amaranthus viridis , Cardamine bonariensis , Chamaesyce hirta , Conyza bonariensis , Digitaria sanguinalis , Gomphrena globosa , Helianthus annuus , Lupinus polyphyllus , Mirabilis jalapa , Oxalis latifolia , Portulaca oleracea and Catharanthus roseus . The phylogenetic analyses of the CSVd variants identified herein showed three groups with Brazilian CSVd variants distributed in them all, which suggests that Brazilian CSVd isolates may have different origins through successive introductions of infected germplasm of chrysanthemum in Brazil.

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.

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.

With increasing interest in RNA biology and the use of RNA molecules in sophisticated biotechnological applications, the methods to produce large amounts of recombinant RNAs are limited. Here, we describe a protocol to produce large amounts of recombinant RNA in Escherichia coli based on co-expression of a chimeric molecule that contains the RNA of interest in a viroid scaffold and a plant tRNA ligase. Viroids are relatively small, non-coding, highly base-paired circular RNAs that are infectious to higher plants. The host plant tRNA ligase is an enzyme recruited by viroids that belong to the family Avsunviroidae, such as Eggplant latent viroid (ELVd), to mediate RNA circularization during viroid replication. Although ELVd does not replicate in E. coli, an ELVd precursor is efficiently transcribed by the E. coli RNA polymerase and processed by the embedded hammerhead ribozymes in bacterial cells, and the resulting monomers are circularized by the co-expressed tRNA ligase reaching a remarkable concentration. The insertion of an RNA of interest into the ELVd scaffold enables the production of tens of milligrams of the recombinant RNA per liter of bacterial culture in regular laboratory conditions. A main fraction of the RNA product is circular, a feature that facilitates the purification of the recombinant RNA to virtual homogeneity. In this protocol, a complementary DNA (cDNA) corresponding to the RNA of interest is inserted in a particular position of the ELVd cDNA in an expression plasmid that is used, along the plasmid to co-express eggplant tRNA ligase, to transform E. coli. Co-expression of both molecules under the control of strong constitutive promoters leads to production of large amounts of the recombinant RNA. The recombinant RNA can be extracted from the bacterial cells and separated from the bulk of bacterial RNAs taking advantage of its circularity.

Due to autotrophic growing capacity and extremely rich secondary metabolism, plants should be preferred targets of synthetic biology. However, developments in plants usually run below those in other taxonomic groups. In this work we engineered genetic circuits capable of logic YES, OR and AND Boolean computation in plant tissues with a visual output signal. The circuits, which are deployed by means of Agrobacterium tumefaciens, perform with the conditional activity of the MYB transcription factor Rosea1 from Antirrhinum majus inducing the accumulation of anthocyanins, plant endogenous pigments that are directly visible to the naked eye or accurately quantifiable by spectrophotometric analysis. The translational fusion of Rosea1 to several viral proteins, such as potyvirus NIb or fragments thereof, rendered the transcription factor inactive. However, anthocyanin accumulation could be restored by inserting protease cleavage sites between both moieties of the fusion and by coexpressing specific proteases, such as potyvirus nuclear inclusion a protease.

Eggplant latent viroid (ELVd) is a relatively small non-coding circular RNA that induces asymptomatic infections in eggplants ( Solanum melongena L.). Like other viroid species that belong to the family Avsunviroidae , ELVd contains hammerhead ribozymes in the strands of both polarities that self-cleave RNAs producing terminal 5'-hydroxyl and 2',3'-cyclic phosphodiester groups. Available experimental data indicate that ELVd replicates in the chloroplasts of infected cells through a symmetric rolling-circle mechanism, in which RNA circularization is catalyzed by the chloroplastic isoform of the tRNA ligase. In this work, a mutational analysis was performed to gain insight into the sequence and structural requirements of the tRNA ligase-mediated circularization of ELVd RNAs. In the predicted minimum free energy conformation of the monomeric linear ELVd RNA intermediate of plus (+) polarity, the ligation site is located in the lower part of an opened internal loop, which is present in a quasi -rod-like structure that occupies the center of the molecule. The mutations analyzed herein consisted of punctual nucleotide substitutions and deletions surrounding the ligation site on the upper and lower strands of the ELVd quasi -double-stranded structure. Computational predictions of the mutated ELVd conformations indicated different degrees of distortions compared to the minimum free energy conformation of the wild-type ELVd linear monomer of + polarity. When these mutant RNAs were expressed in Escherichia coli , they were all circularized by the eggplant tRNA ligase with approximately the same efficiency as the wild-type ELVd, except for those that directly affected the ribozyme domain. These results suggest that the viroid ribozyme domains, in addition to self-cleavage, are also involved in the tRNA ligase-mediated circularization of the monomeric linear replication intermediates.