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.     

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.     

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.     

The molecular structure of hop latent viroid (HLV), a new viroid occurring worldwide in hops.

A new viroid which does not seem to produce any symptoms of disease, and is therefore tentatively named hop latent viroid (HLV) was found to occur worldwide in hops. HLV proved to be infectious when mechanically inoculated onto viroid- and virus-free hops. The viroid nature of HLV was also substantiated by sequence analysis which revealed that HLV is a circular RNA consisting of 256 nucleotides, that can be arranged into the viroid-specific, rod-like secondary structure. HLV also contains the central conserved region typical for most of the presently known viroids. However HLV does not contain the viroid-specific oligo(A) stretch in the upper left part of its rod-like molecule. Because of this feature and a sequence similarity with the prototypes of the other viroid groups below 55%, HLV can be regarded as the first member of a new viroid group.     

Site-specific mutagenesis of potato spindle tuber viroid cDNA:

Summary The infectivity of cloned viroid cDNAs permits investigation of structure/function relationships in these unusual pathogenic RNAs by systematic site-specific mutagenesis of the cDNAs and subsequent bioassay. We have used three different strategies to create nucleotide substitutions within premelting region 2, a region of potato spindle tuber viroid (PSTV) believed to be important in viroid replication: sodium bisulfitecatalyzed deamination of deoxycytosine residues, oligonucleotide-directed mutagenesis, and construction of chimeric viroid cDNAs from fragments of infectious PSTV and tomato apical stunt viroid cDNAs. Although their effects upon the rod-like native structure of PSTV should be minimal, C U transitions at positions 92 or 284 appeared to be lethal. When inoculation with PSTV cDNA containing a single nucleotide substitution was mediated by the Ti plasmid of Agrobacterium tumefaciens, PSTV progeny with an unaltered wild type sequence was obtained. Two factors, the high error frequency characteristic of RNA synthesis and the use of a systemic bioassay for PSTV replication, may explain such sequence reversion and emphasize the importance of an appropriate bioassay system for screening mutant viroid cDNAs.     

Advantages and disadvantages of using PCR techniques to characterize transgenic plants

The polymerase chain reaction (PCR) revolutionized molecular biology to a similar extent as the discovery of plasmids and restriction endonucleases. However, there are some limitations to the use of PCR. Transgenic plants containing potato spindle tuber viroid (PSTVd) cDNA constructs, demonstrated to become de novo methylated upon PSTVd infection, represent a good example to illustrate the advantages of PCR. PSTVd is a 359 nt long autonomously replicating plant pathogenic RNA where all of its enzymatic requirements are entirely provided by the host cell. In addition, viroids that propagate without a DNA intermediate barely tolerate nucleotide substitutions of their RNA genome without losing infectivity. PCR is the method of choice to characterize the sequence context of genome-integrated viroid cDNA or of reverse transcribed PSTVd RNA, and can hardly be replaced by any alternative procedure. Furthermore, the precise examination of DNA methylation patterns (genomic sequencing) is entirely dependent on PCR. In contrast, the use of PCR is critical for the determination of copy number and arrangement of transgene constructs. Here, the advantages and disadvantages of PCR are discussed and protocols for PCR amplification of cDNA, genomic DNA, and bisulfite-treated DNA from transgenic plants are presented.     

Movement of potato spindle tuber viroid reveals regulatory points of phloem-mediated RNA traffic

Increasing evidence indicates that the phloem mediates traffic of selective RNAs within a plant. How an RNA enters, moves in, and exits the phloem is poorly understood. Potato spindle tuber viroid (PSTVd) is a pathogenic RNA that does not encode proteins and is not encapsidated, and yet it replicates autonomously and traffics systemically within an infected plant. The viroid RNA genome must interact directly with cellular factors to accomplish these functions and is, therefore, an excellent probe to study mechanisms that regulate RNA traffic. Our analyses of PSTVd traffic in Nicotiana benthamiana yielded evidence that PSTVd movement within sieve tubes does not simply follow mass flow from source to sink organs. Rather, this RNA is transported into selective sink organs. Furthermore, two PSTVd mutants can enter the phloem to spread systemically but cannot exit the phloem in systemic leaves of tobacco (Nicotiana tabacum). A viroid most likely has evolved structural motifs that mimic endogenous plant RNA motifs so that they are recognized by cellular factors for traffic. Thus, analysis of PSTVd traffic functions may provide insights about endogenous mechanisms that control phloem entry, transport, and exit of RNAs.     

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 human hepatitis delta virus RNAs to dicer activity

The endonuclease dicer cleaves RNAs that are 100% double stranded and certain RNAs with extensive but <100% pairing to release approximately 21-nucleotide (nt) fragments. Circular 1,679-nt genomic and antigenomic RNAs of human hepatitis delta virus (HDV) can fold into a rod-like structure with 74% pairing. However, during HDV replication in hepatocytes of human, woodchuck, and mouse origin, no approximately 21-nt RNAs were detected. Likewise, in vitro, purified recombinant dicer gave <0.2% cleavage of unit-length HDV RNAs. Similarly, rod-like RNAs of potato spindle tuber viroid (PSTVd) and avocado sunblotch viroid (ASBVd) were only 0.5% cleaved. Furthermore, when a 66-nt hairpin RNA with 79% pairing, the putative precursor to miR-122, which is an abundant liver micro-RNA, replaced one end of HDV genomic RNA, it was poorly cleaved, both in vivo and in vitro. In contrast, this 66-nt hairpin, in the absence of appended HDV sequences, was >80% cleaved in vitro. Other 66-nt hairpins derived from one end of genomic HDV, PSTVd, or ASBVd RNAs were also cleaved. Apparently, for unit-length RNAs of HDV, PSTVd, and ASBVd, it is the extended structure with <100% base pairing that confers significant resistance to dicer action.     

Characterization of the RNA motif responsible for the specific interaction of potato spindle tuber viroid RNA (PSTVd) and the tomato protein Virp1

Viroids are small non-coding parasitic RNAs that are able to infect their host plants systemically. This circular naked RNA makes use of host proteins to accomplish its proliferation. Here we analyze the specific binding of the tomato protein Virp1 to the terminal right domain of potato spindle tuber viroid RNA (PSTVd). We find that two asymmetric internal loops within the PSTVd (+) RNA, each composed of the sequence elements 5′-ACAGG and CUCUUCC-5′, are responsible for the specific RNA–protein interaction. In view of the nucleotide composition we call this structural element an ‘RY motif’. The RY motif located close to the terminal right hairpin loop of the PSTVd secondary structure has an ~5-fold stronger binding affinity than the more centrally located RY motif. Simultaneous sequence alterations in both RY motifs abolished the specific binding to Virp1. Mutations in any of the two RY motifs resulted in non-infectious viroid RNA, with the exception of one case, where reversion to sequence wild type took place. In contrast, the simultaneous exchange of two nucleotides within the terminal right hairpin loop of PSTVd had only moderate influence on the binding to Virp1. This variant was infectious and sequence changes were maintained in the progeny. The relevance of the phylogenetic conservation of the RY motif, and sequence elements therein, amongst various genera of the family Pospiviroidae is discussed.