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

A chloroplast protein binds a viroid RNA in vivo and facilitates its hammerhead-mediated self-cleavage

Final full-text version of the paper available at http://www.nature.com/emboj/index.htmlViroids, small single-stranded circular RNAs (246–401 nucleotides), do not have mRNA capacity and must recruit host proteins to assist in the steps of their biological cycle. The nature of these cellular factors is poorly understood due to a lack of reliable experimental approaches. Here, to screen for host proteins interacting with viroid RNAs in vivo, we UV-irradiated avocado leaves infected with avocado sunblotch viroid (ASBVd), the type member of chloroplast viroids containing hammerhead ribozymes. This resulted in the detection of several ASBVd–host protein adducts. Tandem mass spectrometry analysis of the most abundant cross-linked species identified the protein component as two closely related chloroplast RNA-binding proteins (PARBP33 and PARBP35) of a family whose members previously have been shown to be involved in stabilization, maturation and editing of chloroplast transcripts. PARBP33 behaves as an RNA chaperone that stimulates in vitro the hammerhead-mediated self-cleavage of the multimeric ASBVd transcripts that result from rolling circle replication, indicating that this reaction, despite its RNA-based mechanism, is facilitated by proteins. The structural and functional parallelism between PARBP33 and PARBP35, and some proteins involved in viral RNA replication, indicates that viroids and RNA viruses recruit similar host proteins for their replication.R.F. was partially supported by grant PB98-0500 of the DGES de España. J.A.D. was the recipient of a postdoctoral contract from the Ministerio de Ciencia y Tecnología de España.Peer reviewe

Transient expression systems to rewire plant carotenoid metabolism

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.Peer reviewe

Vector para la coexpresión de varias proteínas heterólogas en cantidades equimolares

La invención se refiere a un vector de expresión basado en la secuencia nucleotídica del genoma de un Potyvirus, preferiblemente del virus del grabado del tabaco, que alberga una secuencia nucleotídica que codifica para al menos una proteína heteróloga, preferiblemente para dos y más preferiblemente para tres proteínas heterólogas. Las proteínas heterólogas se expresan, en la célula transfectada con este vector, como parte de la poliproteína viral y se encuentran flanqueadas por secuencias de reconocimiento de la proteasa viral NlaPro, la cual es capaz de liberarlas de la poli proteína completa durante el procesamiento postraduccional. Por tanto, el vector de la invención es útil para la coexpresión de una o de varias proteínas heterólogas en cantidades equimolares en la misma localización subcelular. La invención también se refiere a la célula, preferiblemente vegetal, que comprende este vector de expresión, al polen y a la planta que comprenden esta célula, al germoplasma de esta planta ya los métodos para la obtención de al menos una, dos o tres proteínas heterólogas.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad Politécnica de ValenciaA1 Solicitud de patente con informe sobre el estado de la técnic

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

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

Recombinant RNA production

This invention relates to the production of RNA by co-expressing a tRNA ligase and a chimeric RNA molecule comprising a target RNA and a plant viroid scaffold, such as Eggplant latent viroid, in a host cell. This co-expression causes the production of large amounts of the chimeric RNA molecule in the host cells and may be useful for example in the production of RNA aptamers and other RNA molecules.Peer reviewedConsejo Superior de Investigaciones Científicas (España), Universidad Politécnica de ValenciaA1 Solicitud de patente con informe sobre el estado de la técnic

Proteome expansion in the Potyviridae evolutionary radiation

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.Peer reviewe

HC-Pro hypo- and hypersuppressor mutants: differences in viral siRNA accumulation in vivo and siRNA binding activity in vitro

Viruses have evolved mechanisms to suppress the RNA silencing defense of their hosts, allowing replication and systemic colonization. In a recent study, we found that the effect of mutations in the RNA silencing suppressor of Tobacco etch virus (TEV) was variable, ranging from complete abolition of suppressor activity to significantly stronger suppression. Whereas hyposuppressor mutants were less virulent and accumulated less viral particles than wildtype, hypersuppressors induced symptoms similar to wildtype and accumulated to similar levels. Here we further characterize a set of these mutants in terms of their ability to bind in vitro and induce accumulation in vivo of virus-derived siRNAs. Hyposuppressor alleles are less efficient binding siRNAs than hypersuppressors, whereas the latter are not different from the wildtype. As a consequence of lower viral accumulation, plants infected with virus bearing a hyposuppressor allele also accumulate less virus-derived siRNAs.This work was supported by grants from the Spanish Ministerio de Ciencia e Innovación (MICINN)-Fondo Europeo de Desarrollo Regional (BFU2006-14819-C02-01/BMC), the Generalitat Valenciana (ACOMP07-263) and the European Molecular Biology Organization Young Investigator Program to S.F.E and by grant BIO2008-01986 (MICINN) to J.A.D. C.T.B. was supported by a predoctoral fellowship from the Generalitat Valenciana.Peer reviewe

Reduced Graphene Oxide Nanosheet-Decorated Copper Oxide Nanoparticles: A Potent Antifungal Nanocomposite against Fusarium Root Rot and Wilt Diseases of Tomato and Pepper Plants

© 2020 by the authorsSustainable 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.The APC was funded by the Spanish Ministerio de Ciencia e Innovación (co-financed European Union ERDF), grant number BIO2017-83184-R.Peer reviewe

Method for the production of DSRNA

[EN] The present invention relates to an isolated nucleotide acid sequence comprising a) the cDNAs of two strands of a target double-stranded RNA (dsRNA) separated by an autocatalytic intron flanked by exon fragments, and b) a plant viroid, wherein element (a) is inserted in the plant viroid sequence. The expression of this nucleotide sequence in a host cell, such as E. coli, along with a tRNA ligase, allows the production of high amounts of dsRNA specific for a target gene. This dsRNA can be used in the interfering RNA technology for silencing gene expression. Additionally, the present invention also relates to a method for producing said dsRNA[FR] La présente invention concerne une séquence d'acide nucléotidique isolée comprenant a) les ADNc de deux brins d'un ARN double brin cible (ARNdb)) séparés par un intron autocatalytique flanqué de fragments d'exon, et b) un viroïde végétal, l'élément (a) étant inséré dans la séquence viroïde végétale. L'expression de cette séquence nucléotidique dans une cellule hôte, telle que E. coli, conjointement avec une ligase d'ARNt, permet la production de quantités élevées d'ARNdb spécifique pour un gène cible. Cet ARNdb peut être utilisé dans la technologie d'ARN interférant pour le silençage de l'expression génique. De plus, la présente invention concerne également un procédé de production dudit ARNdbPeer reviewedConsejo Superior de Investigaciones Científicas (España), Universitat Politècnica de ValènciaA1 Solicitud de patente con informe sobre el estado de la técnic