Eggplant latent viroid: a friendly experimental system in the family Avsunviroidae

[EN] Taxonomy Eggplant latent viroid (ELVd) is the only species of the genus Elaviroid (family Avsunviroidae). All the viroids in the family Avsunviroidae contain hammerhead ribozymes in the strands of both polarities, and are considered to replicate in the chloroplasts of infected cells. This family includes two other genera: Avsunviroid and Pelamoviroid. Physical properties ELVd consists of a single-stranded, circular, non-coding RNA of 332–335 nucleotides that folds in a branched quasi-rod-like minimum free-energy conformation. RNAs of complementary polarity exist in infected cells and are considered to be replication intermediates. Plus (+) polarity is assigned arbitrarily to the strand that accumulates at a higher concentration in infected tissues. Host To date, ELVd has only been shown to infect eggplant (Solanum melongena L.), the species in which it was discovered. A very narrow host range seems to be a common property in members of the family Avsunviroidae. Symptoms ELVd infections of eggplants are apparently symptomless. Transmission ELVd is transmitted mechanically and by seed. Useful website http://subviral.med.uottawa.ca.I thank Gustavo G. Gomez [Instituto de Biologia Molecular y Celular de Plantas (IBMCP), Valencia, Spain] for Fig. 4 which illustrates ELVd trafficking into chloroplasts. This work was supported by grants AGL2013-49919-EXP and BIO2014-54269-R from the Spanish Ministerio de Economia y Competitividad (MINECO). The author declares no conflict of interest.Daros Arnau, JA. (2016). Eggplant latent viroid: a friendly experimental system in the family Avsunviroidae. Molecular Plant Pathology. https://doi.org/10.1111/mpp.12358

Tobacco etch virus protein P1 traffics to the nucleolus and associates with the host 60S ribosomal subunits during infection

[EN] The genus Potyvirus comprises a large group of positive-strand RNA plant viruses whose genome encodes a large polyprotein processed by three viral proteinases. P1 protein, the most amino-terminal product of the polyprotein, is an accessory factor stimulating viral genome amplification whose role during infection is not well understood. We infected plants with Tobacco etch virus (TEV; genus Potyvirus) clones in which P1 was tagged with a fluorescent protein to track its expression and subcellular localization or with an affinity tag to identify host proteins involved in complexes in which P1 also takes part during infection. Our results showed that TEV P1 exclusively accumulates in infected cells at an early stage of infection and that the protein displays a dynamic subcellular localization, trafficking in and out of the nucleus and nucleolus during infection. Inside the nucleolus, P1 particularly targets the dense granular component. Consistently, we found functional nucleolar localization and nuclear export signals in TEV P1 sequence. Our results also indicated that TEV P1 physically interacts with the host 80S cytoplasmic ribosomes and specifically binds to the 60S ribosomal subunits during infection. In vitro translation assays of reporter proteins suggested that TEV P1 stimulates protein translation, particularly when driven from the TEV internal ribosome entry site. These in vitro assays also suggested that TEV helper-component proteinase (HC-Pro) inhibits protein translation. Based on these findings, we propose that TEV P1 stimulates translation of viral proteins in infected cells. IMPORTANCE In this work, we researched the role during infection of tobacco etch virus P1 protease. P1 is the most mysterious protein of potyviruses, a relevant group of RNA viruses infecting plants. Our experiments showed that the viral P1 protein exclusively accumulates in infected cells at an early stage of infection and moves in and out of the nucleus of infected cells, particularly targeting the nucleolus. Our experiments also showed that P1 protein binds host ribosomes during infection. Based on these findings and other in vitro experiments we propose that P1 protein stimulates translation of viral proteins during infectionThis work was supported by grant BIO2011-26741 from the Spanish Ministerio de Economia y Competitividad. F.M. was the recipient of a predoctoral fellowship from Universidad Politecnica de Valencia.Martínez, F.; Daros Arnau, JA. (2014). Tobacco etch virus protein P1 traffics to the nucleolus and associates with the host 60S ribosomal subunits during infection. Journal of Virology. 88(18):10725-10737. https://doi.org/10.1128/JVI.00928-14S10725107378818Riechmann, J. L., Lain, S., & Garcia, J. A. (1992). Highlights and prospects of potyvirus molecular biology. 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Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli

[EN] 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 coexpress 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.This work was supported by grants BIO2017-83184-R and BIO2017-91865-EXP from the Spanish Ministerio de Ciencia, Innovacion y Universidades (co-financed FEDER funds).Cordero-Cucart, MT.; Aragonés, V.; Daros Arnau, JA. (2018). Large-scale Production of Recombinant RNAs on a Circular Scaffold Using a Viroid-derived System in Escherichia coli. Journal of Visualized Experiments. (141). https://doi.org/10.3791/58472S14

Virus-host interactome: Putting the accent on how it changes

[EN] Viral infections are extremely complex processes that could only be well understood by precisely characterizing the interaction networks between the virus and the host components. In recent years, much effort has gone in this directionwith the aimof unveiling themolecular basis of viral pathology. These networks are mostly formed by viral and host proteins, and are expected to be dynamic bothwith time and space (i.e., with the progression of infection, as well as with the virus and host genotypes; what we call plastodynamic). This largely overlooked spatio-temporal evolution urgently calls for a change both in the conceptual paradigms and experimental techniques used so far to characterize virus-host interactions. More generally, molecular plasticity and temporal dynamics are unavoidable components of themechanisms that underlie any complex disease; components whose understandingwill eventually enhance our ability to modulate those networkswith the aimof improving disease treatments.This work is supported by the grants BFU2015-66894-P (to G.R.), BI02014-54269-R (to J-A.D.) and BFU2015-65037-P (to S.F.E.) from the Ministerio de Economia, Industria y Competitividad, and by the grant PROMETEOII/2014/021 from the Generalitat Valenciana (to S.F.E. and J-A.D.).Rodrigo Tarrega, G.; Daros Arnau, JA.; Elena Fito, SF. (2017). Virus-host interactome: Putting the accent on how it changes. Journal of Proteomics. 156:1-4. https://doi.org/10.1016/j.jprot.2016.12.007S1415

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

[EN] 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 analyse 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 (a), the replication rate (r) and the degradation rate (d) of viral strands. Our results indicate that the bifurcation associated with a 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. secondorder), absorbing-state phase transition. Finally, we also analyse a simplified model that only incorporates asymmetry in replication tied to differential replication modes.This work was funded by the Human Frontier Science Program Organization grant RGP12/2008, by the Spanish Ministerio de Ciencia e Innovacion grants BIO2008-01986 (J.A.D.) and BFU2009-06993 (S.F.E.) and by the Santa Fe Institute. F. M. is the recipient of a predoctoral fellowship from Universitat Politecnica de Valencia. We also 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).Sardanyes Cayuela, J.; Martinez, F.; Daros Arnau, JA.; Elena Fito, SF. (2012). Dynamics of alternative modes of RNA replication for positive-sense RNA viruses. 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Fast-forward generation of effective artificial small RNAs for enhanced antiviral defense in plants

[EN] Artificial small RNAs (sRNAs) are short ≈21-nt non-coding RNAs engineered to inactivate sequence complementary RNAs. In plants, they have been extensively used to silence cellular transcripts in gene function analyses and to target invading RNA viruses to induce resistance. Current artificial sRNA-based antiviral resistance in plants is mainly limited to a single virus, and is jeopardized by the emergence of mutations in the artificial sRNA target site or by the presence of co-infecting viruses. Hence, there is a need to further develop the artificial sRNA approach to generate more broad and durable antiviral resistance in plants. A recently developed toolbox allows for the time and cost-effective large-scale production of artificial sRNA constructs in plants. The toolbox includes the P-SAMS web tool for the automated design of artificial sRNAs, and a new generation of artificial microRNA and synthetic trans-acting small interfering RNA (syn-tasiRNA) vectors for direct cloning and high expression of artificial sRNAs. Here we describe how the simplicity and high-throughput capability of these new technologies should accelerate the study of artificial sRNA-based antiviral resistance in plants. In particular, we discuss the potential of the syn-tasiRNA approach as a promising strategy for developing more effective, durable and broad antiviral resistance in plants.This study was supported by grants BIO2014-54269-R from Ministerio de Economía y Competitividad (MINECO, Spain) and AI043288 from the U.S. National Institutes of Health. Alberto Carbonell was the recipient of a Marie Sklodowska Curie Individual Fellowship (H2020-MSCA-IF-2014-655841) from the European Commission.Carbonell, A.; Carrington, JC.; Daros Arnau, JA. (2016). Fast-forward generation of effective artificial small RNAs for enhanced antiviral defense in plants. http://www.smartscitech.com/index.php/RD. 3:1-4. https://doi.org/10.14800/rd.1130S14

A viroid-derived system to produce large amounts of recombinant RNA in Escherichia coli

[EN] Viruses have been engineered into useful biotechnological tools for gene therapy or to induce the synthesis of products of interest, such as therapeutic proteins and vaccines, in animal and fungal cells, bacteria or plants. Viroids are a particular class of infectious agents of higher plants that exclusively consist of a small non-protein-coding circular RNA molecule. In the same way as viruses have been transformed into useful biotechnological devices, can viroids be converted into beneficial tools? We show herein that, by expressing Eggplant latent viroid (ELVd) derived RNAs in Escherichia coli together with the eggplant tRNA ligase, this being the enzyme involved in viroid circularization in the infected plant, RNAs of interest like aptamers, extended hairpins, or other structured RNAs are produced in amounts of tens of milligrams per liter of culture. Although ELVd fails to replicate in E. coli, ELVd precursors self-cleave through the embedded hammerhead ribozymes and the resulting monomers are, in part, circularized by the co-expressed enzyme. The mature viroid forms and the protein likely form a ribonucleoprotein complex that transitorily accumulates in E. coli cells at extraordinarily amounts.This work was supported by grant BIO2014-54269-R and BIO2017-83184-R from the Spanish Ministerio de Economia, Industria y Competitividad (co-financed FEDER funds).Daros Arnau, JA.; Aragones, V.; Cordero-Cucart, MT. (2018). A viroid-derived system to produce large amounts of recombinant RNA in Escherichia coli. Scientific Reports. 8:1-9. https://doi.org/10.1038/s41598-018-20314-3S198Di Serio, F. et al. Current status of viroid taxonomy. Arch. Virol. 159, 3467–3478 (2014).Branch, A. D. & Robertson, H. D. A replication cycle for viroids and other small infectious RNAs. Science 223, 450–455 (1984).Branch, A. D., Benenfeld, B. J. & Robertson, H. D. Evidence for a single rolling circle in the replication of potato spindle tuber viroid. Proc. Natl. Acad. Sci. USA 85, 9128–9132 (1988).Daròs, J. A., Marcos, J. F., Hernández, C. & Flores, R. Replication of avocado sunblotch viroid: evidence for a symmetric pathway with two rolling circles and hammerhead ribozyme processing. Proc. Natl. Acad. Sci. USA 91, 12813–12817 (1994).Mühlbach, H. P. & Sänger, H. L. Viroid replication is inhibited by a-amanitin. Nature 278, 185–188 (1979).Navarro, J. A., Vera, A. & Flores, R. A chloroplastic RNA polymerase resistant to tagetitoxin is involved in replication of avocado sunblotch viroid. Virology 268, 218–225 (2000).Gas, M. E., Molina-Serrano, D., Hernández, C., Flores, R. & Daròs, J. A. Monomeric linear RNA of Citrus exocortis viroid resulting from processing in vivo has 5′-phosphomonoester and 3′-hydroxyl termini: implications for the RNase and RNA ligase involved in replication. J. Virol. 82, 10321–10325 (2008).Nohales, M. A., Flores, R. & Daròs, J. A. Viroid RNA redirects host DNA ligase 1 to act as an RNA ligase. Proc. Natl. Acad. Sci. USA 109, 13805–13810 (2012).Nohales, M. A., Molina-Serrano, D., Flores, R. & Daròs, J. A. Involvement of the chloroplastic isoform of tRNA ligase in the replication of viroids belonging to the family Avsunviroidae. J. Virol 86, 8269–8276 (2012).Forster, A. C. & Symons, R. H. Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites. Cell 49, 211–220 (1987).Forster, A. C., Davies, C., Sheldon, C. C., Jeffries, A. C. & Symons, R. H. Self-cleaving viroid and newt RNAs may only be active as dimers. Nature 334, 265–267 (1988).Daròs, J. A. Eggplant latent viroid: a friendly experimental system in the family. Avsunviroidae. Mol. Plant Pathol. 17, 1170–1177 (2016).Paige, J. S., Wu, K. Y. & Jaffrey, S. R. RNA mimics of green fluorescent protein. Science 333, 642–646 (2011).Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806–811 (1998).Zetsche, B. et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).Englert, M. et al. Plant pre-tRNA splicing enzymes are targeted to multiple cellular compartments. Biochimie 89, 1351–1365 (2007).Côté, F. & Perreault, J. P. Peach latent mosaic viroid is locked by a 2′,5′-phosphodiester bond produced by in vitro self-ligation. J. Mol. Biol. 273, 533–543 (1997).Côté, F., Lévesque, D. & Perreault, J. P. Natural 2′,5′-phosphodiester bonds found at the ligation sites of peach latent mosaic viroid. J. Virol. 75, 19–25 (2001).Shaner, N. C. et al. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572 (2004).Martínez, F., Marqués, J., Salvador, M. L. & Daròs, J. A. Mutational analysis of eggplant latent viroid RNA processing in Chlamydomonas reinhardtii chloroplast. J. Gen. Virol. 90, 3057–3065 (2009).Timmons, L., Court, D. L. & Fire, A. Ingestion of bacterially expressed dsRNAs can produce specific and potent genetic interference in Caenorhabditis elegans. Gene 263, 103–112 (2001).Ponchon, L. & Dardel, F. Recombinant RNA technology: the tRNA scaffold. Nat. Methods 4, 571–576 (2007).Ponchon, L. et al. Co-expression of RNA-protein complexes in Escherichia coli and applications to RNA biology. Nucleic Acids Res 41, e150 (2013).Batey, R. T. Advances in methods for native expression and purification of RNA for structural studies. Curr. Opin. Struct. Biol. 26C, 1–8 (2014).Diener, T. O. Viroids: “living fossils” of primordial RNAs? Biol. Direct 11 (2016).Navarro, B. et al. Viroids: How to infect a host and cause disease without encoding proteins. Biochimie 94, 1474–1480 (2012).Engler, C. & Marillonnet, S. Golden Gate cloning. Methods Mol. 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Model-based design of RNA hybridization networks implemented in living cells

[EN] Synthetic gene circuits allow the behavior of living cells to be reprogrammed, and non-coding small RNAs (sRNAs) are increasingly being used as programmable regulators of gene expression. However, sRNAs (natural or synthetic) are generally used to regulate single target genes, while complex dynamic behaviors would require networks of sRNAs regulating each other. Here, we report a strategy for implementing such networks that exploits hybridization reactions carried out exclusively by multifaceted sRNAs that are both targets of and triggers for other sRNAs. These networks are ultimately coupled to the control of gene expression. We relied on a thermo-dynamic model of the different stable conformational states underlying this system at the nucleotide level. To test our model, we designed five different RNA hybridization networks with a linear architecture, and we implemented them in Escherichia coli. We validated the network architecture at the molecular level by native polyacrylamide gel electrophoresis, as well as the network function at the bacterial population and single-cell levels with a fluorescent reporter. Our results suggest that it is possible to engineer complex cellular programs based on RNA from first principles. Because these networks are mainly based on physical interactions, our designs could be expanded to other organisms as portable regulatory resources or to implement biological computations.The Consejo Superior de Investigaciones Cientificas (CSIC) Intramural [grant number 201440I017]; the Ministerio de Economia, Industria y Competitividad (MINECO)/FEDER [grant number BFU2015-66894-P]; and the AXA Research Fund Postdoctoral fellowship to G.R. The predoctoral fellowship [grant number AP2012-3751, MECD] to E.M. The Ministerio de Economia, Industria y Competitividad (MINECO) [grant numbers BIO2014-54269-R, AGL2013-49919-EXP] to J.A.D. The 7th Framework Programme [grant numbers 610730 (EVO-PROG), 613745 (PROMYS)]; the Horizon 2020 Marie Sklodowska-Curie [grant number 642738 (MetaRNA)]; the Engineering and Physical Sciences Research Council (EPSRC) and the Biotechnology and Biological Sciences Research Council (BBSRC) [grant number BB/M017982/1 (WISB centre)]; and the School of Life Sciences (U. Warwick) [startup allocation] to A.J. Funding for open access charge: EPSRC/BBSRC [BB/M017982/1 to A.J.].Rodrigo, G.; Prakash, S.; Shen, S.; Majer, E.; Daros Arnau, JA.; Jaramillo, A. (2017). Model-based design of RNA hybridization networks implemented in living cells. Nucleic Acids Research. 45(16):9797-9808. https://doi.org/10.1093/nar/gkx698S979798084516Ausländer, S., Ausländer, D., Müller, M., Wieland, M., & Fussenegger, M. (2012). Programmable single-cell mammalian biocomputers. Nature, 487(7405), 123-127. doi:10.1038/nature11149Friedland, A. E., Lu, T. K., Wang, X., Shi, D., Church, G., & Collins, J. J. (2009). 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Experimental evolution of pseudogenization and gene loss in a plant RNA virus

[EN] Viruses have evolved highly streamlined genomes and a variety of mechanisms to compress them, suggesting that genome size is under strong selection. Horizontal gene transfer has, on the other hand, played an important role in virus evolution. However, evolution cannot integrate initially nonfunctional sequences into the viral genome if they are rapidly purged by selection. Here we report on the experimental evolution of pseudogenization in virus genomes using a plant RNA virus expressing a heterologous gene. When long 9-week passages were performed, the added gene was lost in all lineages, whereas viruses with large genomic deletions were fixed in only two out of ten 3-week lineages and none in 1-week lineages. Illumina next-generation sequencing revealed considerable convergent evolution in the 9- and 3-week lineages with genomic deletions. Genome size was correlated to within-host competitive fitness, although there was no correlation with virus accumulation or virulence. Within-host competitive fitness of the 3-week virus lineages without genomic deletions was higher than for the 1-week lineages. Our results show that the strength of selection for a reduced genome size and the rate of pseudogenization depend on demographic conditions. Moreover, for the 3-week passage condition, we observed increases in within-host fitness, whereas selection was not strong enough to quickly remove the nonfunctional heterologous gene. These results suggest a demographically determined "sweet spot" might exist, where heterologous insertions are not immediately lost while evolution can act to integrate them into the viral genome.The authors thank Alejandro Manzano Marin for his bioinformatics guidance with the Illumina analysis and Francisca de la Iglesia, Paula Agudo, and Angels Prosper for technical support. This project was made possible through the support of grant 22371 from the John Templeton Foundation to S. F. E. The opinions expressed in this publication are those of the authors and do not necessarily reflect the views of John Templeton Foundation. Additional support was received from the Spanish Direccion General de Investigacion Cientifica y Tecnica grants BFU2012-30805 to S. F. E, JCI2011-10379 to M.P.Z, and BIO2011-26741 to J.A.D., and by a Rubicon grant from the Netherlands Organization for Scientific Research (www.nwo.nl) to M.P.Z.Zwart, MP.; Willemsen, A.; Daros Arnau, JA.; Elena Fito, SF. (2014). Experimental evolution of pseudogenization and gene loss in a plant RNA virus. Molecular Biology and Evolution. 31(1):121-134. https://doi.org/10.1093/molbev/mst175S12113431

Dicer-like 4 is involved in restricting the systemic movement of Zucchini yellow mosaic virus in Nicotiana benthamiana

[EN] Zucchini yellow mosaic virus (ZYMV) induces serious diseases in cucurbits. To create a tool to screen for resistance genes, we cloned a wild ZYMV isolate and inserted the visual marker Roseal to obtain recombinant clone ZYMV-Rosl. While in some plant-virus combinations Roseal induces accumulation of anthocyanins in infected tissues, ZYMV-Rosl infection of cucurbits did not lead to detectable anthocyanin accumulation. However, the recombinant virus did induce dark red pigmen-tation in infected tissues of the model plant Nicotiana ben-thamiana. In this species, ZYMV-Rosl multiplied efficiently in local inoculated tissue but only a few progeny particles estab-lished infection foci in upper leaves. We used this system to analyze the roles of Dicer-like (DCL) genes, core components of plant antiviral RNA silencing pathways, in ZYMV infection. ZYMV-Rosl local replication was not significantly affected in single DCL knockdown lines nor in double DCL2/4 and triple DCL2/3/4 knockdown lines. ZYMV-Rosl systemic accumula-tion was not affected in knockdown lines DCL1, DCL2, and DCL3. However in DCL4 and also in DCL2/4 and DCL2/3/4 knockdown lines, ZYMV-Rosl systemic accumulation dra-matically increased, which highlights the key role of DCL4 in restricting virus systemic movement. The effect of DCL4 on ZYMV systemic movement was confirmed with a wild-type version of the virus.We thank V. Aragones for excellent technical assistance. This work was supported by the Spanish Ministerio de Economia y Competitividad (MINECO) through grants BI02014-54269-R and AGL2013-49919-EXP and by the Greek Ministry for Education and Religious Affairs (Program Aristeia II, 4499, ViroidmiR; ESPA 2007-2013). A. Carbonell was supported by an Individual Fellowship from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement No. 655841.Cordero-Cucart, MT.; Cerdán García, L.; Carbonell Olivares, A.; Katsarou, K.; Kalantidis, K.; Daros Arnau, JA. (2016). Dicer-like 4 is involved in restricting the systemic movement of Zucchini yellow mosaic virus in Nicotiana benthamiana. Molecular Plant-Microbe Interactions. 30(1):63-71. https://doi.org/10.1094/MPMI-11-16-0239-RS637130