Transient expression systems to rewire plant carotenoid metabolism

[EN] 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.The authors are supported by grants BIO2017-83184-R, BIO2017-84041-P, PID2020-114691RB-I00 and PID2020-115810GB-I00 from Spanish MCIN/AEI (10.13039/501100011033) and European Union NextGeneration EU/PRTR. Funding is also provided by the EU PRIMA project UToPIQ (MCIN/AEI code PCI2021-121941), the Generalitat Valenciana PROMETEO project ENIgMA (PROMETEU/2021/056) and the Consejo Superior de Investigaciones Cientificas (CSIC code 202040E299).Rodríguez -Concepción, M.; Daròs, J. (2022). Transient expression systems to rewire plant carotenoid metabolism. Current Opinion in Plant Biology. 66:1-8. https://doi.org/10.1016/j.pbi.2022.102190186

Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses

[EN] 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.We are grateful to Catherine Mark for editorial assistance. We apologize to all colleagues whose relevant publications have not been cited because of space constraints or involuntary forgetfulness. F.P. is supported by a postdoctoral fellowship from the Academia Sinica (Taiwan); J.-A.D. is funded by grant BIO2017-83184-R from the Ministerio de Ciencia, Innovacion y Universidades (Spain). The authors declare no conflict of interest.Pasin, F.; Menzel, W.; Daròs, J. (2019). Harnessed viruses in the age of metagenomics and synthetic biology: an update on infectious clone assembly and biotechnologies of plant viruses. Plant Biotechnology Journal. 17(6):1010-1026. https://doi.org/10.1111/pbi.13084S1010102617

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

[EN] 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.We thank V. Aragones and E. Moya for invaluable technical assistance. This work was supported by grants from Ministerio de Ciencia, Innovacion y Universidades (MCIU, Spain), Agencia Estatal de Investigacion (AEI, Spain) and Fondo Europeo de Desarrollo Regional (FEDER, European Union) (RTI2018-095118-A-100 and RYC-2017-21648 to A.C.; BIO2017-83184-R to J.-A.D.).Carbonell, A.; Lisón, P.; Daròs, J. (2019). Multi-targeting of viral RNAs with synthetic trans-acting small interfering RNAs enhances plant antiviral resistance. 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Molecular Plant Pathology, 18(5), 746-753. doi:10.1111/mpp.12529Carbonell, A., & Daròs, J.-A. (2019). Design, Synthesis, and Functional Analysis of Highly Specific Artificial Small RNAs with Antiviral Activity in Plants. Antiviral Resistance in Plants, 231-246. doi:10.1007/978-1-4939-9635-3_13Carbonell, A., Takeda, A., Fahlgren, N., Johnson, S. C., Cuperus, J. T., & Carrington, J. C. (2014). New Generation of Artificial MicroRNA and Synthetic Trans-Acting Small Interfering RNA Vectors for Efficient Gene Silencing in Arabidopsis. Plant Physiology, 165(1), 15-29. doi:10.1104/pp.113.234989Carbonell, A., Fahlgren, N., Mitchell, S., Cox, K. L., Reilly, K. C., Mockler, T. C., & Carrington, J. C. (2015). Highly specific gene silencing in a monocot species by artificial micro RNA s derived from chimeric mi RNA precursors. The Plant Journal, 82(6), 1061-1075. doi:10.1111/tpj.12835Carbonell, A., López, C., & Daròs, J.-A. (2019). 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Poliovirus Escape from RNA Interference: Short Interfering RNA-Target Recognition and Implications for Therapeutic Approaches. Journal of Virology, 79(2), 1027-1035. doi:10.1128/jvi.79.2.1027-1035.2005Khan, M. Z., Amin, I., Hameed, A., & Mansoor, S. (2018). CRISPR–Cas13a: Prospects for Plant Virus Resistance. Trends in Biotechnology, 36(12), 1207-1210. doi:10.1016/j.tibtech.2018.05.005Khan, M. Z., Haider, S., Mansoor, S., & Amin, I. (2019). Targeting Plant ssDNA Viruses with Engineered Miniature CRISPR-Cas14a. Trends in Biotechnology, 37(8), 800-804. doi:10.1016/j.tibtech.2019.03.015Kis, A., Tholt, G., Ivanics, M., Várallyay, É., Jenes, B., & Havelda, Z. (2015). Polycistronic artificial miRNA-mediated resistance toWheat dwarf virusin barley is highly efficient at low temperature. Molecular Plant Pathology, 17(3), 427-437. doi:10.1111/mpp.12291KUNG, Y.-J., LIN, S.-S., HUANG, Y.-L., CHEN, T.-C., HARISH, S. S., CHUA, N.-H., & YEH, S.-D. (2011). Multiple artificial microRNAs targeting conserved motifs of the replicase gene confer robust transgenic resistance to negative-sense single-stranded RNA plant virus. Molecular Plant Pathology, 13(3), 303-317. doi:10.1111/j.1364-3703.2011.00747.xLafforgue, G., Martinez, F., Sardanyes, J., de la Iglesia, F., Niu, Q.-W., Lin, S.-S., … Elena, S. F. (2011). Tempo and Mode of Plant RNA Virus Escape from RNA Interference-Mediated Resistance. Journal of Virology, 85(19), 9686-9695. doi:10.1128/jvi.05326-11Lafforgue, G., Martinez, F., Niu, Q.-W., Chua, N.-H., Daros, J.-A., & Elena, S. F. (2013). Improving the Effectiveness of Artificial MicroRNA (amiR)-Mediated Resistance against Turnip Mosaic Virus by Combining Two amiRs or by Targeting Highly Conserved Viral Genomic Regions. Journal of Virology, 87(14), 8254-8256. doi:10.1128/jvi.00914-13Levanova, A., & Poranen, M. M. (2018). RNA Interference as a Prospective Tool for the Control of Human Viral Infections. Frontiers in Microbiology, 9. doi:10.3389/fmicb.2018.02151Lin, S.-S., Wu, H.-W., Elena, S. F., Chen, K.-C., Niu, Q.-W., Yeh, S.-D., … Chua, N.-H. (2009). Molecular Evolution of a Viral Non-Coding Sequence under the Selective Pressure of amiRNA-Mediated Silencing. PLoS Pathogens, 5(2), e1000312. doi:10.1371/journal.ppat.1000312Liu, Q., Wang, F., & Axtell, M. J. (2014). Analysis of Complementarity Requirements for Plant MicroRNA Targeting Using a Nicotiana benthamiana Quantitative Transient Assay  . The Plant Cell, 26(2), 741-753. doi:10.1105/tpc.113.120972Llave, C., Xie, Z., Kasschau, K. D., & Carrington, J. C. (2002). Cleavage of Scarecrow-like mRNA Targets Directed by a Class of Arabidopsis miRNA. Science, 297(5589), 2053-2056. doi:10.1126/science.1076311Mahas, A., & Mahfouz, M. (2018). Engineering virus resistance via CRISPR–Cas systems. Current Opinion in Virology, 32, 1-8. doi:10.1016/j.coviro.2018.06.002Martínez, F., Lafforgue, G., Morelli, M. J., González-Candelas, F., Chua, N.-H., Daròs, J.-A., & Elena, S. F. (2012). Ultradeep Sequencing Analysis of Population Dynamics of Virus Escape Mutants in RNAi-Mediated Resistant Plants. Molecular Biology and Evolution, 29(11), 3297-3307. doi:10.1093/molbev/mss135Mehta, D., Stürchler, A., Anjanappa, R. B., Zaidi, S. S.-A., Hirsch-Hoffmann, M., Gruissem, W., & Vanderschuren, H. (2019). Linking CRISPR-Cas9 interference in cassava to the evolution of editing-resistant geminiviruses. Genome Biology, 20(1). doi:10.1186/s13059-019-1678-3Mitter, N., Zhai, Y., Bai, A. X., Chua, K., Eid, S., Constantin, M., … Pappu, H. R. (2016). Evaluation and identification of candidate genes for artificial microRNA-mediated resistance to tomato spotted wilt virus. Virus Research, 211, 151-158. doi:10.1016/j.virusres.2015.10.003Montgomery, T. A., Howell, M. D., Cuperus, J. T., Li, D., Hansen, J. E., Alexander, A. L., … Carrington, J. C. (2008). 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Simplifying plant gene silencing and genome editing logistics by a one-Agrobacterium system for simultaneous delivery of multipartite virus vectors

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

Processing of Nuclear Viroids In Vivo: An Interplay between RNA Conformations

Replication of viroids, small non-protein-coding plant pathogenic RNAs, entails reiterative transcription of their incoming single-stranded circular genomes, to which the (+) polarity is arbitrarily assigned, cleavage of the oligomeric strands of one or both polarities to unit-length, and ligation to circular RNAs. While cleavage in chloroplastic viroids (family Avsunviroidae) is mediated by hammerhead ribozymes, where and how cleavage of oligomeric (+) RNAs of nuclear viroids (family Pospiviroidae) occurs in vivo remains controversial. Previous in vitro data indicated that a hairpin capped by a GAAA tetraloop is the RNA motif directing cleavage and a loop E motif ligation. Here we have re-examined this question in vivo, taking advantage of earlier findings showing that dimeric viroid (+) RNAs of the family Pospiviroidae transgenically expressed in Arabidopsis thaliana are processed correctly. Using this methodology, we have mapped the processing site of three members of this family at equivalent positions of the hairpin I/double-stranded structure that the upper strand and flanking nucleotides of the central conserved region (CCR) can form. More specifically, from the effects of 16 mutations on Citrus exocortis viroid expressed transgenically in A. thaliana, we conclude that the substrate for in vivo cleavage is the conserved double-stranded structure, with hairpin I potentially facilitating the adoption of this structure, whereas ligation is determined by loop E and flanking nucleotides of the two CCR strands. These results have deep implications on the underlying mechanism of both processing reactions, which are most likely catalyzed by enzymes different from those generally assumed: cleavage by a member of the RNase III family, and ligation by an RNA ligase distinct from the only one characterized so far in plants, thus predicting the existence of at least a second plant RNA ligase

Intron-assisted, viroid-based production of insecticidal circular double-stranded RNA in Escherichia coli

[EN] 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.This work was supported by the Ministerio de Ciencia e Innovacion (Spain; co-financed by the European Regional Development Fund) [BIO2017-83184-R] and [BIO2017-91865-EXP]; Universitat Politecnica de Valencia [PAID-01-17]. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).Ortolá-Navarro, B.; Cordero, T.; Hu, X.; Daròs, J. (2021). Intron-assisted, viroid-based production of insecticidal circular double-stranded RNA in Escherichia coli. RNA Biology. 18(11):1846-1857. https://doi.org/10.1080/15476286.2021.1872962S18461857181

Dynamics of alternative modes of RNA replication for positive-sense RNA viruses

We propose and study nonlinear mathematical models describing the intracellular time dynamics of viral RNA accumulation for positive sense single-stranded RNA viruses. Our models consider different replication modes ranging between two extremes represented by the geometric replication (GR) and the linear stamping machine replication (SMR). We first analyze a model that quantitatively reproduced experimental data for the accumulation dynamics of both polarities of Turnip mosaic potyvirus RNAs. We identify a non-degenerate transcritical bifurcation governing the extinction of both strands depending on three key parameters: the mode of replication (®), the replication rate (r) and the degradation rate (±) of viral strands. Our results indicate that the bifurcation associated with ® generically takes place when the replication mode is closer to the SMR, thus suggesting that GR may provide viral strands with an increased robustness against degradation. This transcritical bifurcation, which is responsible for the switching from an active to an absorbing regime, suggests a smooth (i.e., second-order), absorbing-state phase transition. Finally, we also analyze a simplified model that only incorporates asymmetry in replication tied to differential replication modes.This work has been funded by the Human Frontier Science Program Organization grant RGP12/2008, by the Spanish Ministerio de Ciencia e Innovaci´on grants BIO2008-01986 (JAD) and BFU2009-06993 (SFE), and by the Santa Fe Institute. FM is the recipient of a predoctoral fellowship from Universitat Polit`ecnica de Val`encia. We also want to thank the hospitality and support of the Kavli Institute for Theoretical Physics (University of California at Santa Barbara), where part of this work was developed (grant NSF PHY05-51164).Peer reviewe

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

[EN] 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.Ministerio de Ciencia, Innovacion y Universidades (MCIU); Agencia Estatal de Investigacion (AEI); Fondo Europeo de Desarrollo Regional (FEDER) [RTI2018-095118-A-100; RYC-2017-21648 to A.C.; BIO2017-83184-R to J.-A.D.]. Funding for open access charge: MCIU; AEI; FEDER [RTI2018-095118-A-100].López-Dolz, L.; Spada, M.; Daròs, J.; Carbonell, A. (2020). Fine-tune control of targeted RNAi efficacy by plant artificial small RNAs. Nucleic Acids Research. 48(11):6234-6250. https://doi.org/10.1093/nar/gkaa343S62346250481

CRISPR-Cas12a genome editing at the whole-plant level using two compatible RNA virus vectors

[EN] 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.This research was supported by grants BIO2017-83184-R and PID2019-108203RB-I00 from the Ministerio de Ciencia e Innovacion (Spain) through the Agencia Estatal de Investigacion (co-financed by the European Regional Development Fund) and H2020-760331 Newcotiana from the European Commission. M.U. andM.V.-V. are the recipients of fellowships FPU17/05503 from the Ministerio de Ciencia e Innovacion (Spain) and APOSTD/2020/096 from the Generalitat Valenciana (Spain), respectivelyUranga, M.; Vázquez-Vilar, M.; Orzáez Calatayud, DV.; Daròs, J. (2021). CRISPR-Cas12a genome editing at the whole-plant level using two compatible RNA virus vectors. The CRISPR Journal. 4(5):1-9. https://doi.org/10.1089/crispr.2021.0049194

Evolutionary Constraints to Viroid Evolution

We suggest that viroids are trapped into adaptive peaks as the result of adaptive constraints. The first one is imposed by the necessity to fold into packed structures to escape from RNA silencing. This creates antagonistic epistases, which make future adaptive trajectories contingent upon the first mutation and slow down the rate of adaptation. This second constraint can only be surpassed by increasing genetic redundancy or by recombination. Eigen’s paradox imposes a limit to the increase in genome complexity in the absence of mechanisms reducing mutation rate. Therefore, recombination appears as the only possible route to evolutionary innovation in viroids