Effective Antiviral Application of Antisense in Plants by Exploiting Accessible Sites in the Target RNA.

Antisense oligodeoxynucleotides (ASOs) have long been used to selectively inhibit or modulate gene expression at the RNA level, and some ASOs are approved for clinical use. However, the practicability of antisense technologies remains limited by the difficulty of reliably predicting the sites accessible to ASOs in complex folded RNAs. Recently, we applied a plant-based method that reproduces RNA-induced RNA silencing in vitro to reliably identify sites in target RNAs that are accessible to small interfering RNA (siRNA)-guided Argonaute endonucleases. Here, we show that this method is also suitable for identifying ASOs that are effective in DNA-induced RNA silencing by RNases H. We show that ASOs identified in this way that target a viral genome are comparably effective in protecting plants from infection as siRNAs with the corresponding sequence. The antiviral activity of the ASOs could be further enhanced by chemical modification. This led to two important conclusions: siRNAs and ASOs that can effectively knock down complex RNA molecules can be identified using the same approach, and ASOs optimized in this way could find application in crop protection. The technology developed here could be useful not only for effective RNA silencing in plants but also in other organisms.

Expression of symptoms elicited by a hammerhead viroid through RNA silencing is related to population bottlenecks in the infected host.

Chlorosis is frequently incited by viroids, small nonprotein-coding, circular RNAs replicating in nuclei (family Pospiviroidae) or chloroplasts (family Avsunviroidae). Here, we investigated how chrysanthemum chlorotic mottle viroid (CChMVd, Avsunviroidae) colonizes, evolves and initiates disease. Progeny variants of natural and mutated CChMVd sequence variants inoculated in chrysanthemum plants were characterized, and plant responses were assessed by molecular assays. We showed that: chlorotic mottle induced by CChMVd reflects the spatial distribution and evolutionary behaviour in the infected host of pathogenic (containing a UUUC tetranucleotide) and nonpathogenic (lacking such a pathogenic determinant) variants; and RNA silencing is involved in the initiation of the chlorosis in symptomatic leaf sectors through a viroid-derived small RNA containing the pathogenic determinant that directs AGO1-mediated cleavage of the mRNA encoding the chloroplastic transketolase. This study provides the first evidence that colonization of leaf tissues by CChMVd is characterized by segregating variant populations differing in pathogenicity and with the ability to colonize leaf sectors (bottlenecks) and exclude other variants (superinfection exclusion). Importantly, no specific pathogenic viroid variants were found in the chlorotic spots caused by chrysanthemum stunt viroid (Pospiviroidae), thus establishing a clear distinction on how members of the two viroid families trigger chlorosis in the same host.

Altered expression levels of long non-coding natural antisense transcripts overlapping the UGT73C6 gene affect rosette size in Arabidopsis thaliana.

Natural antisense long non-coding RNAs (lncNATs) are involved in the regulation of gene expression in plants, modulating different relevant developmental processes and responses to various stimuli. We have identified and characterized two lncNATs (NAT1UGT73C6 and NAT2UGT73C6 , collectively NATsUGT73C6 ) from Arabidopsis thaliana that are transcribed from a gene fully overlapping UGT73C6, a member of the UGT73C subfamily of genes encoding UDP-glycosyltransferases (UGTs). Expression of both NATsUGT73C6 is developmentally controlled and occurs independently of the transcription of UGT73C6 in cis. Downregulation of NATsUGT73C6 levels through artificial microRNAs results in a reduction of the rosette area, while constitutive overexpression of NAT1UGT73C6 or NAT2UGT73C6 leads to the opposite phenotype, an increase in rosette size. This activity of NATsUGT73C6 relies on its RNA sequence and, although modulation of UGT73C6 in cis cannot be excluded, the observed phenotypes are not a consequence of the regulation of UGT73C6 in trans. The NATsUGT73C6 levels were shown to affect cell proliferation and thus individual leaf size. Consistent with this concept, our data suggest that the NATsUGT73C6 influence the expression levels of key transcription factors involved in regulating leaf growth by modulating cell proliferation. These findings thus reveal an additional regulatory layer on the process of leaf growth. In this work, we characterized at the molecular level two long non-coding RNAs (NATsUGT73C6 ) that are transcribed in the opposite direction to UGT73C6, a gene encoding a glucosyltransferase involved in brassinosteroid homeostasis in A. thaliana. Our results indicate that NATsUGT73C6 expression influences leaf growth by acting in trans and by modulating the levels of transcription factors that are involved in the regulation of cell proliferation.

Role of RNA silencing in plant-viroid interactions and in viroid pathogenesis.

Viroids are small, single-stranded, non-protein coding and circular RNAs able to infect host plants in the absence of any helper virus. They may elicit symptoms in their hosts, but the underlying molecular pathways are only partially known. Here we address the role of post-transcriptional RNA silencing in plant-viroid-interplay, with major emphasis on the involvement of this sequence-specific RNA degradation mechanism in both plant antiviroid defence and viroid pathogenesis. This review is a tribute to the memory of Dr. Ricardo Flores, who largely contributed to elucidate this and other molecular mechanisms involved in plant-viroid interactions.

RNA and Protein Determinants Mediate Differential Binding of miRNAs by a Viral Suppressor of RNA Silencing Thus Modulating Antiviral Immune Responses in Plants.

Many plant viruses express suppressor proteins (VSRs) that can inhibit RNA silencing, a central component of antiviral plant immunity. The most common activity of VSRs is the high-affinity binding of virus-derived siRNAs and thus their sequestration from the silencing process. Since siRNAs share large homologies with miRNAs, VSRs like the Tombusvirus p19 may also bind miRNAs and in this way modulate cellular gene expression at the post-transcriptional level. Interestingly, the binding affinity of p19 varies considerably between different miRNAs, and the molecular determinants affecting this property have not yet been adequately characterized. Addressing this, we analyzed the binding of p19 to the miRNAs 162 and 168, which regulate the expression of the important RNA silencing constituents Dicer-like 1 (DCL1) and Argonaute 1 (AGO1), respectively. p19 binds miRNA162 with similar high affinity as siRNA, whereas the affinity for miRNA168 is significantly lower. We show that specific molecular features, such as mismatches and 'G-U wobbles' on the RNA side and defined amino acid residues on the VSR side, mediate this property. Our observations highlight the remarkable adaptation of VSR binding affinities to achieve differential effects on host miRNA activities. Moreover, they show that even minimal changes, i.e., a single base pair in a miRNA duplex, can have significant effects on the efficiency of the plant antiviral immune response.

A life of research on circular RNAs and ribozymes: towards the origin of viroids, deltaviruses and life.

The first examples of circular RNAs (circRNAs) were reported in the '70s as a family of minimal infectious agents of flowering plants; the viroids and viral satellites of circRNA. In some cases, these small circular genomes encode self-cleaving RNA motifs or ribozymes, including an exceptional circRNA infecting not plants but humans: the Hepatitis Delta Virus. Autocatalytic ribozymes not only allowed to propose a common rolling-circle replication mechanism for all these subviral agents, but also a tentative link with the origin of life as molecular fossils of the so-called RNA world. Despite the weak biologic connection between angiosperm plants and the human liver, diverse scientists, and most notably Ricardo Flores, firmly supported an evolutionary relationship between plant viroids and human deltavirus agents. The tireless and inspiring work done by Ricardo's lab in the field of infectious circRNAs fuelled multiple hypotheses for the origin of these entities, allowing advances in other fields, from eukaryotic circRNAs to small ribozymes in genomes from all life kingdoms. The recent discovery of a plethora of viral-like circRNAs with ribozymes in disparate biological samples may finally allow us to connect plant and animal subviral agents, confirming again that Ricardo's eye for science was always a keen eye.

Highly efficacious antiviral protection of plants by small interfering RNAs identified in vitro.

In response to a viral infection, the plant's RNA silencing machinery processes viral RNAs into a huge number of small interfering RNAs (siRNAs). However, a very few of these siRNAs actually interfere with viral replication. A reliable approach to identify these immunologically effective siRNAs (esiRNAs) and to define the characteristics underlying their activity has not been available so far. Here, we develop a novel screening approach that enables a rapid functional identification of antiviral esiRNAs. Tests on the efficacy of such identified esiRNAs of a model virus achieved a virtual full protection of plants against a massive subsequent infection in transient applications. We find that the functionality of esiRNAs depends crucially on two properties: the binding affinity to Argonaute proteins and the ability to access the target RNA. The ability to rapidly identify functional esiRNAs could be of great benefit for all RNA silencing-based plant protection measures against viruses and other pathogens.

Different rates of spontaneous mutation of chloroplastic and nuclear viroids as determined by high-fidelity ultra-deep sequencing.

Mutation rates vary by orders of magnitude across biological systems, being higher for simpler genomes. The simplest known genomes correspond to viroids, subviral plant replicons constituted by circular non-coding RNAs of few hundred bases. Previous work has revealed an extremely high mutation rate for chrysanthemum chlorotic mottle viroid, a chloroplast-replicating viroid. However, whether this is a general feature of viroids remains unclear. Here, we have used high-fidelity ultra-deep sequencing to determine the mutation rate in a common host (eggplant) of two viroids, each representative of one family: the chloroplastic eggplant latent viroid (ELVd, Avsunviroidae) and the nuclear potato spindle tuber viroid (PSTVd, Pospiviroidae). This revealed higher mutation frequencies in ELVd than in PSTVd, as well as marked differences in the types of mutations produced. Rates of spontaneous mutation, quantified in vivo using the lethal mutation method, ranged from 1/1000 to 1/800 for ELVd and from 1/7000 to 1/3800 for PSTVd depending on sequencing run. These results suggest that extremely high mutability is a common feature of chloroplastic viroids, whereas the mutation rates of PSTVd and potentially other nuclear viroids appear significantly lower and closer to those of some RNA viruses.

ICTV Virus Taxonomy Profile: Ophioviridae.

The Ophioviridae is a family of filamentous plant viruses, with single-stranded negative, and possibly ambisense, RNA genomes of 11.3-12.5 kb divided into 3-4 segments, each encapsidated separately. Virions are naked filamentous nucleocapsids, forming kinked circles of at least two different contour lengths. The sole genus, Ophiovirus, includes seven species. Four ophioviruses are soil-transmitted and their natural hosts include trees, shrubs, vegetables and bulbous or corm-forming ornamentals, both monocots and dicots. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Ophioviridae, which is available at http://www.ictv.global/report/ophioviridae.

Viroids, infectious long non-coding RNAs with autonomous replication.

Transcriptome deep-sequencing studies performed during the last years confirmed that the vast majority of the RNAs transcribed in higher organisms correspond to several types of non-coding RNAs including long non-coding RNAs (lncRNAs). The study of lncRNAs and the identification of their functions, is still an emerging field in plants but the characterization of some of them indicate that they play an important role in crucial regulatory processes like flowering regulation, and responses to abiotic stress and plant hormones. A second group of lncRNAs present in plants is formed by viroids, exogenous infectious subviral plant pathogens well known since many years. Viroids are composed of circular RNA genomes without protein-coding capacity and subvert enzymatic activities of their hosts to complete its own biological cycle. Different aspects of viroid biology and viroid-host interactions have been elucidated in the last years and some of them are the main topic of this review together with the analysis of the state-of-the-art about the growing field of endogenous lncRNAs in plants.

The transcription initiation sites of eggplant latent viroid strands map within distinct motifs in their in vivo RNA conformations.

Eggplant latent viroid (ELVd), like other members of family Avsunviroidae, replicates in plastids through a symmetric rolling-circle mechanism in which elongation of RNA strands is most likely catalyzed by a nuclear-encoded polymerase (NEP) translocated to plastids. Here we have addressed where NEP initiates transcription of viroid strands. Because this step is presumably directed by sequence/structural motifs, we have previously determined the conformation of the monomeric linear (+) and (-) RNAs of ELVd resulting from hammerhead-mediated self-cleavage. In silico predictions with 3 softwares led to similar bifurcated conformations for both ELVd strands. In vitro examination by non-denaturing PAGE showed that they migrate as prominent single bands, with the ELVd (+) RNA displaying a more compact conformation as revealed by its faster electrophoretic mobility. In vitro SHAPE analysis corroborated the ELVd conformations derived from thermodynamics-based predictions in silico. Moreover, sequence analysis of 94 full-length natural ELVd variants disclosed co-variations, and mutations converting canonical into wobble pairs or vice versa, which confirmed in vivo most of the stems predicted in silico and in vitro, and additionally helped to introduce minor structural refinements. Therefore, results from the 3 experimental approaches were essentially consistent among themselves. Application to RNA preparations from ELVd-infected tissue of RNA ligase-mediated rapid amplification of cDNA ends, combined with pretreatments to modify the 5' ends of viroid strands, mapped the transcription initiation sites of ELVd (+) and (-) strands in vivo at different sequence/structural motifs, in contrast with the situation previously observed in 2 other members of the family Avsunviroidae.

Viroids: survivors from the RNA world?

Because RNA can be a carrier of genetic information and a biocatalyst, there is a consensus that it emerged before DNA and proteins, which eventually assumed these roles and relegated RNA to intermediate functions. If such a scenario--the so-called RNA world--existed, we might hope to find its relics in our present world. The properties of viroids that make them candidates for being survivors of the RNA world include those expected for primitive RNA replicons: (a) small size imposed by error-prone replication, (b) high G + C content to increase replication fidelity, (c) circular structure for assuring complete replication without genomic tags, (d) structural periodicity for modular assembly into enlarged genomes, (e) lack of protein-coding ability consistent with a ribosome-free habitat, and (f) replication mediated in some by ribozymes, the fingerprint of the RNA world. With the advent of DNA and proteins, those protoviroids lost some abilities and became the plant parasites we now know.

The Arabidopsis tandem zinc finger 9 protein binds RNA and mediates pathogen-associated molecular pattern-triggered immune responses.

Recognition of pathogen-associated molecular patterns (PAMPs) induces multiple defense mechanisms to limit pathogen growth. Here, we show that the Arabidopsis thaliana tandem zinc finger protein 9 (TZF9) is phosphorylated by PAMP-responsive mitogen-activated protein kinases (MAPKs) and is required to trigger a full PAMP-triggered immune response. Analysis of a tzf9 mutant revealed attenuation in specific PAMP-triggered reactions such as reactive oxygen species accumulation, MAPK activation and, partially, the expression of several PAMP-responsive genes. In accordance with these weaker PAMP-triggered responses, tzf9 mutant plants exhibit enhanced susceptibility to virulent Pseudomonas syringae pv. tomato DC3000. Visualization of TZF9 localization by fusion to green fluorescent protein revealed cytoplasmic foci that co-localize with marker proteins of processing bodies (P-bodies). This localization pattern is affected by inhibitor treatments that limit mRNA availability (such as cycloheximide or actinomycin D) or block nuclear export (leptomycin B). Coupled with its ability to bind the ribohomopolymers poly(rU) and poly(rG), these results suggest involvement of TZF9 in post-transcriptional regulation, such as mRNA processing or storage pathways, to regulate plant innate immunity.

Population structure of Citrus tristeza virus from field Argentinean isolates.

We studied the genetic variability of three genomic regions (p23, p25 and p27 genes) from 11 field Citrus tristeza virus isolates from the two main citrus growing areas of Argentina, a country where the most efficient vector of the virus, Toxoptera citricida, is present for decades. The pathogenicity of the isolates was determinated by biological indexing, single-strand conformation polymorphism analysis showed that most isolates contained high intra-isolate variability. Divergent sequence variants were detected in some isolates, suggesting re-infections of the field plants. Phylogenetic analysis of the predominant sequence variants of each isolate revealed similar grouping of isolates for genes p25 and p27. The analysis of p23 showed two groups contained the severe isolates. Our results showed a high intra-isolate sequence variability suggesting that re-infections could contribute to the observed variability and that the host can play an important role in the selection of the sequence variants present in these isolates.

Citrus psorosis virus RNA 1 is of negative polarity and potentially encodes in its complementary strand a 24K protein of unknown function and 280K putative RNA dependent RNA polymerase.

Citrus psorosis virus (CPsV), the type member of genus Ophiovirus, has three genomic RNAs. Complete sequencing of CPsV RNA 1 revealed a size of 8184 nucleotides and Northern blot hybridization with chain specific probes showed that its non-coding strand is preferentially encapsidated. The complementary strand of RNA 1 contains two open reading frames (ORFs) separated by a 109-nt intergenic region, one located near the 5'-end potentially encoding a 24K protein of unknown function, and another of 280K containing the core polymerase motifs characteristic of viral RNA-dependent RNA polymerases (RdRp). Comparison of the core RdRp motifs of negative-stranded RNA viruses, supports grouping CPsV, Ranunculus white mottle virus (RWMV) and Mirafiori lettuce virus (MiLV) within the same genus (Ophiovirus), constituting a monophyletic group separated from all other negative-stranded RNA viruses. Furthermore, RNAs 1 of MiLV, CPsV and RWMV are similar in size and those of MiLV and CPsV also in genomic organization and sequence.