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

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. 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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

Carotenoid fortification of zucchini fruits using a viral RNA vector

[EN] 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 ¿- and ß-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. Graphical Abstract and Lay Summary Inoculation of zucchini plants with a zucchini yellow mosaic virus (ZYMV) vector that expresses a bacterial phytoene synthase (crtB) induces yellow-orange fruits that accumulate health-promoting carotenoids. This is an alternative strategy for metabolic fortification of edible fruits that avoids labor-intensive and time-consuming approaches based on breeding or plant transformation.Fakhreddine Houhou, and Maricarmen Martí, contributed equally to the work. The authors thank the gene bank from Instituto Universitario de Conservación y Mejora de la Agrodiversidad Valenciana (COMAV), Universistat Politècnica de València. This research was supported by grants BIO2017-83184-R, BIO2017-84041-P, BIO2017-90877-REDT, AGL2017-85563-C2-1-R, PID2020-114691RB-I00, PID2020-115810GB-I00, and PID2020-116055RB-C21 from Ministerio de Ciencia e Innovación (Spain), through Agencia Estatal de Investigación (cofinanced European Regional Development Fund) Maricarmen Martí is the recipient of a predoctoral fellowship (FPU16/05294) from Ministerio de Educación, Cultura y Deporte (Spain).Fakhreddine-Houhou, H.; Martí-Botella, MC.; Cordero-Cucart, MT.; Aragones, V.; Sáez-Sánchez, C.; Cebolla Cornejo, J.; Pérez De Castro, AM.... (2022). Carotenoid fortification of zucchini fruits using a viral RNA vector. Biotechnology Journal. e2100328:1-10. https://doi.org/10.1002/biot.202100328110e210032