Resistance of tomato infected with cucumber mosaic virus satellite RNA to potato spindle tuber viroid

Tomato (Lycopersicon esculentum cvs Rutgers and Lichun) plants were firstly pre-inoculated either with a cucumber mosaic virus (CMV) isolate containing satellite RNA (CMV-S52) or with a CMV isolate without satellite RNA, and then challenged 14 days later with a severe strain of potato spindle tuber viroid (PSTVd). Also, tomato plants transformed with CMV satellite cDNA and non-transgenic control plants were directly inoculated with PSTVd. Protection effects were assessed by the observation of symptoms and by assay of PSTVd accumulation in tomato plants using return polyacrylamide gel electrophoresis and silver staining. The results indicated that the satellite-transgenic plants and plants pre-inoculated with CMV-S52 showed much milder symptoms of PSTVd infection than the respective control plants. The concentration of PSTVd RNA in the satellite-transgenic plants and CMV-S52 pre-inoculated plants was reduced to about 0.02-0.03 of the controls. PSTVd infection did not increase the amount of satellite ds-RNA in plants. It is concluded that the plant resistance to PSTVd is induced by the presence of satellite RNA rather than the CMV infection. It is suggested that as there is considerable sequence similarity between satellite RNA and PSTVd, base pairings may be a cause of reduction of both symptoms and the accumulation of PSTVd.     

A structured viroid RNA serves as a substrate for dicer-like cleavage to produce biologically active small RNAs but is resistant to RNA-induced silencing complex-mediated degradation

RNA silencing is a potent means of antiviral defense in plants and animals. A hallmark of this defense response is the production of 21- to 24-nucleotide viral small RNAs via mechanisms that remain to be fully understood. Many viruses encode suppressors of RNA silencing, and some viral RNAs function directly as silencing suppressors as counterdefense. The occurrence of viroid-specific small RNAs in infected plants suggests that viroids can trigger RNA silencing in a host, raising the question of how these noncoding and unencapsidated RNAs survive cellular RNA-silencing systems. We address this question by characterizing the production of small RNAs of Potato spindle tuber viroid (srPSTVds) and investigating how PSTVd responds to RNA silencing. Our molecular and biochemical studies provide evidence that srPSTVds were derived mostly from the secondary structure of viroid RNAs. Replication of PSTVd was resistant to RNA silencing, although the srPSTVds were biologically active in guiding RNA-induced silencing complex (RISC)-mediated cleavage, as shown with a sensor system. Further analyses showed that without possessing or triggering silencing suppressor activities, the PSTVd secondary structure played a critical role in resistance to RISC-mediated cleavage. These findings support the hypothesis that some infectious RNAs may have evolved specific secondary structures as an effective means to evade RNA silencing in addition to encoding silencing suppressor activities. Our results should have important implications in further studies on RNA-based mechanisms of host-pathogen interactions and the biological constraints that shape the evolution of infectious RNA structures.     

Resistance of tomato infected with cucumber mosaic virus satellite RNA to potato spindle tuber viroid

Tomato (Lycopersicon esculentum cvs Rutgers and Lichun) plants were firstly pre-inoculated either with a cucumber mosaic virus (CMV) isolate containing satellite RNA (CMV-S52) or with a CMV isolate without satellite RNA, and then challenged 14 days later with a severe strain of potato spindle tuber viroid (PSTVd). Also, tomato plants transformed with CMV satellite cDNA and non-transgenic control plants were directly inoculated with PSTVd. Protection effects were assessed by the observation of symptoms and by assay of PSTVd accumulation in tomato plants using return polyacrylamide gel electrophoresis and silver staining. The results indicated that the satellite-transgenic plants and plants pre-inoculated with CMV-S52 showed much milder symptoms of PSTVd infection than the respective control plants. The concentration of PSTVd RNA in the satellite-transgenic plants and CMV-S52 pre-inoculated plants was reduced to about 0.02–0.03 of the controls. PSTVd infection did not increase the amount of satellite ds-RNA in plants. It is concluded that the plant resistance to PSTVd is induced by the presence of satellite RNA rather than the CMV infection. It is suggested that as there is considerable sequence similarity between satellite RNA and PSTVd, base pairings may be a cause of reduction of both symptoms and the accumulation of PSTVd.     

Scion on a Stock Producing siRNAs of Potato Spindle Tuber Viroid (PSTVd) Attenuates Accumulation of the Viroid

Plants can attenuate the replication of plant viruses and viroids by RNA silencing induced by virus and viroid infection. In higher plants, silencing signals such as small interfering RNAs (siRNAs) produced by RNA silencing can be transported systemically through phloem, so it is anticipated that antiviral siRNA signals produced in a stock would have the potential to attenuate propagation of viruses or viroids in the scion. To test whether this is indeed the case, we prepared transgenic tobacco (Nicotiana benthamiana) expressing a hairpin RNA (hpRNA) of Potato spindle tuber viroid (PSTVd) in companion cells by using a strong companion cell-specific promoter. A grafting experiment of the wild type tobacco scion on the top of the transgenic tobacco stock revealed that accumulation of PSTVd challenge-inoculated into the scion was apparently attenuated compared to the control grafted plants. These results indicate that genetically modified rootstock expressing viroid-specific siRNAs can attenuate viroid accumulation in a non-genetically modified scion grafted on the stock.     

RNA-dependent RNA polymerase 1 delays the accumulation of viroids in infected plants

RNA-dependent RNA polymerase 1 (RDR1) is essential for plant antiviral defence, but its role in plant defence against viroid infection remains unknown. The present study aimed to identify the function and mechanism of RDR1 in plant resistance to viroid infection. Overexpression of Nicotiana tabacum RDR1 (NtRDR1) delayed the accumulation of potato spindle tuber viroid (PSTVd) genomic RNA and PSTVd-derived small RNA (sRNA) in Nicotiana benthamiana plants at the early invasion stage, but not in the late stage of infection. Conversely, virus-induced gene silencing of tomato RDR1 (SlRDR1a) increased the susceptibility to PSTVd infection (increased viroid accumulation). Salicylic acid (SA) pretreatment induced SlRDR1a expression and enhanced the defence against PSTVd infection in tomato plants. Our study demonstrated that RDR1 is involved in SA-mediated defence and restricts the early systemic invasion by PSTVd in plants. The decreased PSTVd accumulation in N. benthamiana was not caused by efficient accumulation of PSTVd sRNAs. These results deepen our understanding of the mechanism of RDR1 in plant defence responses to viroid attack.     

Inhibition of cell growth and shoot development by a specific nucleotide sequence in a noncoding viroid RNA

Viroids are small noncoding and infectious RNAs that replicate autonomously and move systemically throughout an infected plant. The RNAs of the family Pospiviroidae contain a central conserved region (CCR) that has long been thought to be involved in replication. Here, we report that the CCR of Potato spindle tuber viroid (PSTVd) also plays a role in pathogenicity. A U257A change in the CCR converted the intermediate strain PSTVd(Int) to a lethal strain that caused severe growth stunting and premature death of infected plants. PSTVd with nucleotide U257 changed to C or G did not cause such symptoms. The pathogenic effect of the U257A substitution was abolished by a C259U substitution in the same RNA. Analyses of the pathogenic effects of the U257A substitution in three other PSTVd variants established A257 as a new pathogenicity determinant that functions independently and synergistically with the classic pathogenicity domain. The U257A substitution did not alter PSTVd secondary structure, replication levels, or tissue tropism. The stunted growth of PSTVd(Int)U257A-infected tomato plants resulted from restricted cell expansion but not cell division or differentiation. This was correlated positively with the downregulated expression of an expansin gene, LeExp2. Our results demonstrate that specific nucleotides in a noncoding, pathogenic RNA have a profound effect in altering distinct cellular responses, which then lead to well-defined alterations in plant growth and developmental patterns. The feasibility of correlating viroid RNA sequence/structure with the altered expression of specific host genes, cellular processes, and developmental patterns makes viroid infection a valuable system in which to investigate host factors for symptom expression and perhaps also to characterize the mechanisms of RNA regulation of gene expression in plants.     

Virp1 is a host protein with a major role in Potato spindle tuber viroid infection in Nicotiana plants

Viroids are small, circular, single-stranded RNA molecules that, while not coding for any protein, cause several plant diseases. Viroids rely for their infectious cycle on host proteins, most of which are likely to be involved in endogenous RNA-mediated phenomena. Therefore, characterization of host factors interacting with the viroid may contribute to the elucidation of RNA-related pathways of the hosts. Potato spindle tuber viroid (PSTVd) infects several members of the Solanaceae family. In an RNA ligand screening we have previously isolated the tomato protein Virp1 by its ability to specifically interact with PSTVd positive-strand RNA. Virp1 is a bromodomain-containing protein with an atypical RNA binding domain and a nuclear localization signal. Here we investigate the role of Virp1 in the viroid infection cycle by the use of transgenic lines of Nicotiana tabacum and Nicotiana benthamiana that either overexpress the tomato Virp1 RNA or suppress the orthologous Nicotiana genes through RNA silencing. Plants of the Virp1-suppressed lines were not infected by PSTVd or Citrus exocortis viroid through mechanical inoculation, indicating a major role of Virp1 in viroid infection. On the other hand, overexpression of tomato Virp1 in N. tabacum and N. benthamiana plants did not affect PSTVd KF 440-2 infectivity or symptomatology in these species. Transfection experiments with isolated protoplasts revealed that Virp1-suppressed cells were unable to sustain viroid replication, suggesting that resistance to viroid infection in Virp1-suppressed plants is likely the result of cell-autonomous events.     

Global analysis of tomato gene expression during Potato spindle tuber viroid infection reveals a complex array of changes affecting hormone signaling

Viroids like Potato spindle tuber viroid (PSTVd) are the smallest known agents of infectious disease-small, highly structured, circular RNA molecules that lack detectable messenger RNA activity, yet are able to replicate autonomously in susceptible plant species. To better understand the possible role of RNA silencing in disease induction, a combination of microarray analysis and large-scale RNA sequence analysis was used to compare changes in tomato gene expression and microRNA levels associated with PSTVd infection in two tomato cultivars plus a third transformed line expressing small PSTVd small interfering RNAs in the absence of viroid replication. Changes in messenger (m)RNA levels for the sensitive cultivar 'Rutgers' were extensive, involving more than half of the approximately 10,000 genes present on the array. Chloroplast biogenesis was down-regulated in both sensitive and tolerant cultivars, and effects on mRNAs encoding enzymes involved in the biosynthesis of gibberellin and other hormones were accompanied by numerous changes affecting their respective signaling pathways. In the dwarf cultivar 'MicroTom', a marked upregulation of genes involved in response to stress and other stimuli was observed only when exogenous brassinosteroid was applied to infected plants, thereby providing the first evidence for the involvement of brassinosteroid-mediated signaling in viroid disease induction.     

Tertiary structure and function of an RNA motif required for plant vascular entry to initiate systemic trafficking

Vascular entry is a decisive step for the initiation of long-distance movement of infectious and endogenous RNAs, silencing signals and developmental/defense signals in plants. However, the mechanisms remain poorly understood. We used Potato spindle tuber viroid (PSTVd) as a model to investigate the direct role of the RNA itself in vascular entry. We report here the identification of an RNA motif that is required for PSTVd to traffic from nonvascular into the vascular tissue phloem to initiate systemic infection. This motif consists of nucleotides U/C that form a water-inserted cis Watson-Crick/Watson-Crick base pair flanked by short helices that comprise canonical Watson-Crick/Watson-Crick base pairs. This tertiary structural model was inferred by comparison with X-ray crystal structures of similar motifs in rRNAs and is supported by combined mutagenesis and covariation analyses. Hydration pattern analysis suggests that water insertion induces a widened minor groove conducive to protein and/or RNA interactions. Our model and approaches have broad implications to investigate the RNA structural motifs in other RNAs for vascular entry and to study the basic principles of RNA structure-function relationships.     

RNAi-mediated resistance to Potato spindle tuber viroid in transgenic tomato expressing a viroid hairpin RNA construct

Because of their highly ordered structure, mature viroid RNA molecules are assumed to be resistant to degradation by RNA interference (RNAi). In this article, we report that transgenic tomato plants expressing a hairpin RNA (hpRNA) construct derived from Potato spindle tuber viroid (PSTVd) sequences exhibit resistance to PSTVd infection. Resistance seems to be correlated with high-level accumulation of hpRNA-derived short interfering RNAs (siRNAs) in the plant. Thus, although small RNAs produced by infecting viroids [small RNAs of PSTVd (srPSTVds)] do not silence viroid RNAs efficiently to prevent their replication, hpRNA-derived siRNAs (hp-siRNAs) appear to effectively target the mature viroid RNA. Genomic mapping of the hp-siRNAs revealed an unequal distribution of 21- and 24-nucleotide siRNAs of both (+)- and (–)-strand polarities along the PSTVd genome. These data suggest that RNAi can be employed to engineer plants for viroid resistance, as has been well established for viruses.     

A chimeric satellite transgene sequence is inefficiently targeted by viroid-induced DNA methylation in tobacco

In plants, transgenes containing Potato spindle tuber viroid (PSTVd) cDNA sequences were efficient targets of PSTVd infection-mediated RNA-directed DNA methylation. Here, we demonstrate that in PSTVd-infected tobacco plants, a 134 bp PSTVd fragment (PSTVd-134) did not become densely methylated when it was inserted into a chimeric Satellite tobacco mosaic virus (STMV) construct. Only about 4–5% of all cytosines (Cs) of the PSTVd-134 were methylated when flanked by satellite sequences. In the same plants, C methylation was approximately 92% when the PSTVd-134 was in a PSTVd full length sequence context and roughly 33% when flanked at its 3′ end by a 19 bp PSTVd and at its 5′ end by a short viroid-unrelated sequence. In addition, PSTVd small interfering RNAs (siRNAs) produced from the replicating viroid failed to target PSTVd-134-containing chimeric STMV RNA for degradation. Satellite RNAs appear to have adopted secondary structures that protect them against RNA interference (RNAi)—mediated degradation. Protection can be extended to short non-satellite sequences residing in satellite RNAs, rendering them poor targets for nuclear and cytoplasmic RNAi induced in trans.     

Potato spindle tuber viroid: the simplicity paradox resolved?

Taxonomy:   Potato spindle tuber viroid (PSTVd) is the type species of the genus Posipiviroid , family Pospiviroidae . An absence of hammerhead ribozymes and the presence of a 'central conserved region' distinguish PSTVd and related viroids from members of a second viroid family, the Avsunviroidae .
Physical properties:   Viroids are small, unencapsidated, circular, single-stranded RNA molecules which replicate autonomously when inoculated into host plants. Because viroids are non-protein-coding RNAs, designation of the more abundant, highly infectious polarity strand as the positive strand is arbitrary. PSTVd assumes a rod-like, highly structured conformation that is resistant to nuclease degradation in vitro . Naturally occurring sequence variants of PSTVd range in size from 356 to 361 nt.
Hosts and symptoms:   The natural host range of PSTVd—cultivated potato, certain other Solanum spp., and avocado—appears to be quite limited. Foliar symptoms in potato are often obscure, and the severity of tuber symptoms (elongation with the appearance of prominent bud scales/eyebrows and growth cracks) depends on both temperature and length of infection. PSTVd has a broad experimental host range, especially among solanaceous species, and strains are classified as mild, intermediate or severe based upon the symptoms observed in sensitive tomato cultivars. These symptoms include shortening of internodes, petioles and mid-ribs, severe epinasty and wrinkling of the leaves, and necrosis of mid-ribs, petioles and stems.     

Differential subnuclear localization of RNA strands of opposite polarity derived from an autonomously replicating viroid

The wide variety of RNAs produced in the nucleus must be localized correctly to perform their functions. However, the mechanism of this localization is poorly understood. We report here the differential subnuclear localization of RNA strands of opposite polarity derived from the replicating Potato spindle tuber viroid (PSTVd). During replication, (+)- and (-)-strand viroid RNAs are produced. We found that in infected cultured cells and plants, the (-)-strand RNA was localized in the nucleoplasm, whereas the (+)-strand RNA was localized in the nucleolus as well as in the nucleoplasm with distinct spatial patterns. Furthermore, the presence of the (+)-PSTVd in the nucleolus caused the redistribution of a small nucleolar RNA. Our results support a model in which (1) the synthesis of the (-)- and (+)-strands of PSTVd RNAs occurs in the nucleoplasm, (2) the (-)-strand RNA is anchored in the nucleoplasm, and (3) the (+)-strand RNA is transported selectively into the nucleolus. Our results imply that the eukaryotic cell has a machinery that recognizes and localizes the opposite strands of an RNA, which may have broad ramifications in the RNA regulation of gene expression and the infection cycle of pathogenic RNAs and in the development of RNA-based methods to control gene expression as well as pathogen infection.