Effect of VIRP1 Protein on Nuclear Import of Citrus Exocortis Viroid (CEVd).

Before replicating, Pospiviroidae viroids must move into the plant nucleus. However, the mechanisms of viroid nuclear import are not entirely understood. To study the nuclear import of viroids, we established a nuclear import assay system using onion cell strips and observed the import of Alexa Fluor-594-labeled citrus exocortis viroid (CEVd). To identify the plant factors involved in the nuclear import of viroids, we cloned the Viroid RNA-binding Protein 1 (VIRP1) gene from a tomato cultivar, Seokwang, and heterologously expressed and purified the VIRP1 protein. The newly prepared VIRP1 protein had alterations of amino acid residues at two points (H52R, A277G) compared with a reference VIRP1 protein (AJ249595). VIRP1 specifically bound to CEVd and promoted its nuclear import. However, it is still uncertain whether VIRP1 is the only factor required for the nuclear import of CEVd because CEVd entered the plant nuclei without VIRP1 in our assay system. The cause of the observed nuclear accumulation of CEVd in the absence of VIRP1 needs to be further clarified.

Integrated Proteo-Transcriptomic Analyses Reveal Insights into Regulation of Pollen Development Stages and Dynamics of Cellular Response to Apple Fruit Crinkle Viroid (AFCVd)-Infection in Nicotiana tabacum.

Tobacco (Nicotiana tabacum) pollen is a well-suited model for studying many fundamental biological processes owing to its well-defined and distinct development stages. It is also one of the major agents involved in the transmission of infectious viroids, which is the primary mechanism of viroid pathogenicity in plants. However, some viroids are non-transmissible and may be possibly degraded or eliminated during the gradual process of pollen development maturation. The molecular details behind the response of developing pollen against the apple fruit crinkle viroid (AFCVd) infection and viroid eradication is largely unknown. In this study, we performed an integrative analysis of the transcriptome and proteome profiles to disentangle the molecular cascade of events governing the three pollen development stages: early bicellular pollen (stage 3, S3), late bicellular pollen (stage 5, S5), and 6 h-pollen tube (PT6). The integrated analysis delivered the molecular portraits of the developing pollen against AFCVd infection, including mechanistic insights into the viroid eradication during the last steps of pollen development. The isobaric tags for label-free relative quantification (iTRAQ) with digital gene expression (DGE) experiments led us to reliably identify subsets of 5321, 5286, and 6923 proteins and 64,033, 60,597, and 46,640 expressed genes in S3, S5, and PT6, respectively. In these subsets, 2234, 2108 proteins and 9207 and 14,065 mRNAs were differentially expressed in pairwise comparisons of three stages S5 vs. S3 and PT6 vs. S5 of control pollen in tobacco. Correlation analysis between the abundance of differentially expressed mRNAs (DEGs) and differentially expressed proteins (DEPs) in pairwise comparisons of three stages of pollen revealed numerous discordant changes in mRNA/protein pairs. Only a modest correlation was observed, indicative of divergent transcription, and its regulation and importance of post-transcriptional events in the determination of the fate of early and late pollen development in tobacco. The functional and enrichment analysis of correlated DEGs/DEPs revealed the activation in pathways involved in carbohydrate metabolism, amino acid metabolism, lipid metabolism, and cofactor as well as vitamin metabolism, which points to the importance of these metabolic pathways in pollen development. Furthermore, the detailed picture of AFCVd-infected correlated DEGs/DEPs was obtained in pairwise comparisons of three stages of infected pollen. The AFCVd infection caused the modulation of several genes involved in protein degradation, nuclear transport, phytohormone signaling, defense response, and phosphorylation. Intriguingly, we also identified several factors including, DNA-dependent RNA-polymerase, ribosomal protein, Argonaute (AGO) proteins, nucleotide binding proteins, and RNA exonucleases, which may plausibly involve in viroid stabilization and eradication during the last steps of pollen development. The present study provides essential insights into the transcriptional and translational dynamics of tobacco pollen, which further strengthens our understanding of plant-viroid interactions and support for future mechanistic studies directed at delineating the functional role of candidate factors involved in viroid elimination.

Organ-specific differences in endogenous phytohormone and antioxidative responses in potato upon PSTVd infection.

Although structurally simple, viroids can trigger numerous changes in host plants and cause loss of yield in agronomically important crops. This study investigated changes in the endogenous status of phytohormones and antioxidant enzyme activity in Solanum tuberosum cv. Désirée in response to Potato spindle tuber viroid (PSTVd) infection. Phytohormone analysis showed that the content of endogenous jasmonic acid (JA) and its precursor cis-OPDA significantly increased in leaves, while the content of castasterone (CS) increased in both leaves and tubers of systemically infected plants compared to mock-inoculated control plants at 8 weeks post-inoculation. The indole-3-acetic acid content moderately increased only in tubers, while no differences in salicylic acid and abscisic acid content were observed between infected and control plants. Changes in endogenous phytohormone content were associated with upregulated expression of genes involved in the biosynthesis of JA and brassinosteroids, and the metabolism of auxins. Additionally, PSTVd infection provoked overproduction of hydrogen peroxide, which coincided with increased activity of guaiacol peroxidase in leaves and ascorbate peroxidase in potato tubers. The activity of catalase decreased in leaves, while superoxide dismutase activity remained steady regardless of the treatment and organ type. Total ascorbate and glutathione did not change significantly, although a shift towards oxidized forms was observed. Results suggest the existence of organ-specific differences in phytohormone and antioxidative responses in potato upon PSTVd infection. Possible effects of the observed changes on symptom development are discussed.

Allelic RNA Motifs in Regulating Systemic Trafficking of Potato Spindle Tuber Viroid.

Intercellular RNA trafficking has been shown as a widely-existing phenomenon that has significant functions in many aspects of biology. Viroids, circular noncoding RNAs that cause plant diseases, have been a model to dissect the role of RNA structural motifs in regulating intercellular RNA trafficking in plants. Recent studies on potato spindle tuber viroid (PSTVd) showed that the RNA motif loop 19 is important for PSTVd to spread from palisade to spongy mesophyll in infected leaves. Here, we performed saturated mutational analysis to uncover all possible functional variants of loop 19 and exploit this data to pinpoint to a three-dimensional structural model of this motif. Interestingly, we found that two distinct structural motifs can replace loop 19 and retain the systemic trafficking capacity. One of the alternative structures rapidly emerged from the inoculation using a loop 19 abolished mutant that is not capable of systemic trafficking. Our observation indicates the flexibility of multiple structural arrangements interchangeably exerting similar function at a particular RNA locus. Taken together, this study deepens the understanding of RNA structural motifs-regulated viroid RNA trafficking, which has broad implications for studying RNA intercellular trafficking as well.

Changes in the DNA methylation pattern of the host male gametophyte of viroid-infected cucumber plants.

Eukaryotic organisms exposed to adverse conditions are required to show a certain degree of transcriptional plasticity in order to cope successfully with stress. Epigenetic regulation of the genome is a key regulatory mechanism allowing dynamic changes of the transcriptional status of the plant in response to stress. The Hop stunt viroid (HSVd) induces the demethylation of ribosomal RNA (rRNA) in cucumber (Cucumis sativus) leaves, leading to increasing transcription rates of rRNA. In addition to the clear alterations observed in vegetative tissues, HSVd infection is also associated with drastic changes in gametophyte development. To examine the basis of viroid-induced alterations in reproductive tissues, we analysed the cellular and molecular consequences of HSVd infection in the male gametophyte of cucumber plants. Our results indicate that in the pollen grain, accumulation of HSVd RNA induces a decondensation of the generative nucleus that correlates with a dynamic demethylation of repetitive regions in the cucumber genome that include rRNA genes and transposable elements (TEs). We therefore propose that HSVd infection impairs the epigenetic control of rRNA genes and TEs in gametic cells of cucumber, a phenomenon thus far unknown to occur in this reproductive tissue as a consequence of pathogen infection.

Replicating Potato spindle tuber viroid mediates de novo methylation of an intronic viroid sequence but no cleavage of the corresponding pre-mRNA.

In plants, Potato spindle tuber viroid (PSTVd) replication triggers post-transcriptional gene silencing (PTGS) and RNA-directed DNA methylation (RdDM) of homologous RNA and DNA sequences, respectively. PTGS predominantly occurs in the cytoplasm, but nuclear PTGS has been also reported. In this study, we investigated whether the nuclear replicating PSTVd is able to trigger nuclear PTGS. Transgenic tobacco plants carrying cytoplasmic and nuclear PTGS sensor constructs were PSTVd-infected resulting in the generation of abundant PSTVd-derived small interfering RNAs (vd-siRNAs). Northern blot analysis revealed that, in contrast to the cytoplasmic sensor, the nuclear sensor transcript was not targeted for RNA degradation. Bisulfite sequencing analysis showed that the nuclear PTGS sensor transgene was efficiently targeted for RdDM. Our data suggest that PSTVd fails to trigger nuclear PTGS, and that RdDM and nuclear PTGS are not necessarily coupled.

Specific argonautes selectively bind small RNAs derived from potato spindle tuber viroid and attenuate viroid accumulation in vivo.

The identification of viroid-derived small RNAs (vd-sRNAs) of 21 to 24 nucleotides (nt) in plants infected by viroids (infectious non-protein-coding RNAs of just 250 to 400 nt) supports their targeting by Dicer-like enzymes, the first host RNA-silencing barrier. However, whether viroids, like RNA viruses, are also targeted by the RNA-induced silencing complex (RISC) remains controversial. At the RISC core is one Argonaute (AGO) protein that, guided by endogenous or viral sRNAs, targets complementary RNAs. To examine whether AGO proteins also load vd-sRNAs, leaves of Nicotiana benthamiana infected by potato spindle tuber viroid (PSTVd) were agroinfiltrated with plasmids expressing epitope-tagged versions of AGO1, AGO2, AGO3, AGO4, AGO5, AGO6, AGO7, AGO9, and AGO10 from Arabidopsis thaliana. Immunoprecipitation analyses of the agroinfiltrated halos revealed that all AGOs except AGO6, AGO7, and AGO10 associated with vd-sRNAs: AGO1, AGO2, and AGO3 preferentially with those of 21 and 22 nt, while AGO4, AGO5, and AGO9 additionally bound those of 24 nt. Deep-sequencing analyses showed that sorting of vd-sRNAs into AGO1, AGO2, AGO4, and AGO5 depended essentially on their 5'-terminal nucleotides, with the profiles of the corresponding AGO-loaded vd-sRNAs adopting specific hot spot distributions along the viroid genome. Furthermore, agroexpression of AGO1, AGO2, AGO4, and AGO5 on PSTVd-infected tissue attenuated the level of the genomic RNAs, suggesting that they, or their precursors, are RISC targeted. In contrast to RNA viruses, PSTVd infection of N. benthamiana did not affect miR168-mediated regulation of the endogenous AGO1, which loaded vd-sRNAs with specificity similar to that of its A. thaliana counterpart. Importance: To contain invaders, particularly RNA viruses, plants have evolved an RNA-silencing mechanism relying on the generation by Dicer-like (DCL) enzymes of virus-derived small RNAs of 21 to 24 nucleotides (nt) that load and guide Argonaute (AGO) proteins to target and repress viral RNA. Viroids, despite their minimal genomes (non-protein-coding RNAs of only 250 to 400 nt), infect and incite disease in plants. The accumulation in these plants of 21- to 24-nt viroid-derived small RNAs (vd-sRNAs) supports the notion that DCLs also target viroids but does not clarify whether vd-sRNAs activate one or more AGOs. Here, we show that in leaves of Nicotiana benthamiana infected by potato spindle tuber viroid, the endogenous AGO1 and distinct AGOs from Arabidopsis thaliana that were overexpressed were associated with vd-sRNAs displaying the same properties (5'-terminal nucleotide and size) previously established for endogenous and viral small RNAs. Overexpression of AGO1, AGO2, AGO4, and AGO5 attenuated viroid accumulation, supporting their role in antiviroid defense.

Experimental transmission of pospiviroid populations to weed species characteristic of potato and hop fields.

Weed plants characteristic for potato and hop fields have not been considered in the past as potential hosts that could transmit and lead to spreading of potato spindle tuber (PSTVd) and hop stunt (HSVd) viroids, respectively. To gain insight into this problem, we biolistically inoculated these weed plants with viroid populations either as RNA or as cDNA. New potential viroid host species, collected in central Europe, were discovered. From 12 weed species characteristic for potato fields, high viroid levels, detectable by molecular hybridization, were maintained after both RNA and DNA transfers in Chamomilla reculita and Anthemis arvensis. Low viroid levels, detectable by reverse transcription-PCR (RT-PCR) only, were maintained after plant inoculations with cDNA in Veronica argensis and Amaranthus retroflexus. In these two species PSTVd concentrations were 10(5) and 10(3) times, respectively, lower than in tomato as estimated by real-time PCR. From 14 weeds characteristic for hop fields, high HSVd levels were detected in Galinsoga ciliata after both RNA and DNA transfers. HSVd was found, however, not to be transmissible by seeds of this weed species. Traces of HSVd were detectable by RT-PCR in HSVd-cDNA-inoculated Amaranthus retroflexus. Characteristic monomeric (+)-circular and linear viroid RNAs were present in extracts from weed species propagating viroids to high levels, indicating regular replication, processing, and circularization of viroid RNA in these weed species. Sequence analyses of PSTVd progenies propagated in C. reculita and A. arvensis showed a wide spectrum of variants related to various strains, from mild to lethal variants; the sequence variants isolated from A. retroflexus and V. argensis exhibited similarity or identity to the superlethal AS1 viroid variant. All HSVd clones from G. ciliata corresponded to a HSVdg variant, which is strongly pathogenic for European hops.

Tertiary structural and functional analyses of a viroid RNA motif by isostericity matrix and mutagenesis reveal its essential role in replication.

RNA-templated RNA replication is essential for viral or viroid infection, as well as for regulation of cellular gene expression. Specific RNA motifs likely regulate various aspects of this replication. Viroids of the Pospiviroidae family, as represented by the Potato spindle tuber viroid (PSTVd), replicate in the nucleus by utilizing DNA-dependent RNA polymerase II. We investigated the role of the loop E (sarcin/ricin) motif of the PSTVd genomic RNA in replication. A tertiary-structural model of this motif, inferred by comparative sequence analysis and comparison with nuclear magnetic resonance and X-ray crystal structures of loop E motifs in other RNAs, is presented in which core non-Watson-Crick base pairs are precisely specified. Isostericity matrix analysis of these base pairs showed that the model accounts for the reported natural sequence variations and viable experimental mutations in loop E motifs of PSTVd and other viroids. Furthermore, isostericity matrix analysis allowed us to design disruptive, as well as compensatory, mutations of PSTVd loop E. Functional analyses of such mutants by in vitro and in vivo experiments demonstrated that loop E structural integrity is crucial for replication, specifically during transcription. Our results suggest that the PSTVd loop E motif exists and functions in vivo and provide loss-of-function genetic evidence for the essential role of a viroid RNA three-dimensional motif in rolling-circle replication. The use of isostericity matrix analysis of non-Watson-Crick base pairing to rationalize mutagenesis of tertiary motifs and systematic in vitro and in vivo functional assays of mutants offers a novel, comprehensive approach to elucidate the tertiary-structure-function relationships for RNA motifs of general biological significance.

The dimerization domain of potato spindle tuber viroid, a possible hallmark for infectious RNA.

Covalently closed circular (+) RNA of the potato spindle tuber viroid (PSTVd) can efficiently dimerize noncovalently upon heating and slow cooling in the presence of monovalent cations or Mg2+. In vitro transcription of subgenomic fragments reveals that the ability to dimerize resides in the "upper strand" of its self-complementary rod-like structure. Nuclease probing of these fragments, namely, molecules spanning either the upper or the lower strand of PSTVd, confirms the existence of the previously proposed hairpins I-III, of which hairpin I might contain noncanonical G.A and A.A base pairs. In addition, the upper and lower (+) strands contain large hairpin loops consisting of stretches rich in either adenosine or uridine. Dimerization of the upper (+) strand results in a nuclease-resistant core encompassing hairpin I and is inhibited by an antisense oligonucleotide spanning the entire hairpin; this palindromic domain thus represents the dimerization site. When upper and lower strands were heated and cooled together, no annealing to a viroid-like duplex of both molecules occurs, only dimerization of the upper strand. Therefore, the dimerization hairpin of viroid RNA represents a unique conformational signal that is homologous to similar regions in the human immunodeficiency virus and other retroviruses.

Viroid processing: switch from cleavage to ligation is driven by a change from a tetraloop to a loop E conformation.

A longer-than-unit-length transcript of potato spindle tuber viroid is correctly processed in a potato nuclear extract only if the central conserved region is folded into a multi-helix junction containing at least one GNRA tetraloop-hairpin. The cleavage-ligation site between G95 and G96 was mapped with S1 nuclease and primer extension. The structural motifs involved in the processing mechanism were analysed by UV crosslinking, chemical mapping, phylogenetic comparison and thermodynamic calculations. For processing, the first cleavage occurs within the stem of the GNRA tetraloop; a local conformational change switches the tetraloop motif into a loop E motif, stabilizing a base-paired 5' end. The second cleavage yields unit-length linear intermediates, whose 3' end is also base-paired and most probably coaxially stacked in optimum juxtaposition to the 5' end. They are ligated to mature circles autocatalytically, with low efficiency, or enzymatically, with high efficiency.

The gradual reduction of viroid levels in hop mericlones following heat therapy: a possible role for a nuclease degrading dsRNA.

A high concentration of hop latent viroid (HLVd) was detected in infected mericlones of Osvald's hops grown in vitro. This concentration was about 8-fold higher than in leaves of young, field-grown plants, reaching about 30 pg/mg of fresh mass. Treatment of these in vitro-grown plants at high temperature (35 degrees C) for two weeks lead to a dramatic (about 70-90%) decrease of HLVd content. More detailed investigations performed with mericlone 6147 of Osvald 31 showed that HLVd levels decrease gradually during subsequent cycles of heat treatment. A nuclease activity capable of cleaving HLVd and fully double-stranded RNA was shown to increase significantly in hop tissues during thermotherapy cycles, or after the heat shock. The nuclease activity was found to have similar properties to those extracted earlier from tobacco anthers. This enzyme resembles a sugar-unspecific nuclease which has a maximum activity at pH 5.5. Analysis of the activity with viroid and dsRNA showed that both, endo- and exonucleolytic activities were attributable to the enzyme. A strong tissue-specific gradient of viroid (the lowest level in stem apex and the highest level in roots) was observed in young plants, showing a negative correlation with the dsRNAse activity. In senescent plants, the highest viroid concentration was observed in maturated cones and in upper stems. High nuclease activity in the upper stem tissue suggests that viroid RNA must be protected in this tissue against degradation.