Eggplant latent viroid,the candidate type species for a new genus within the family Avsunviroidae (hammerhead viroids)

Viroids, small circular RNAs that replicate independently and in most cases incite diseases in plants, are classified into the families Pospiviroidae, composed of species with a central conserved region (CCR) and without hammerhead ribozymes, and Avsunviroidae, composed of three members lacking CCR but able to self-cleave in both polarity strands through hammerhead ribozymes. Here we report the biological and molecular properties of Eggplant latent viroid (ELVd). Purified circular ELVd induces symptomless infections when inoculated into eggplant seedlings. ELVd can be transmitted horizontally and through seed. Sequencing 10 complete cDNA clones showed that ELVd is a circular RNA of 332 to 335 nucleotides with high variability. This RNA can adopt a quasi-rod-like secondary structure of minimal free energy and alternative foldings that permit formation of stable hammerhead structures in plus and minus strands. The ribozymes are active in vitro and, most likely, in vivo. Considering the ELVd properties to be intermediate between those of the two genera of family Avsunviroidae, we propose ELVd as the type species of a third genus with the name ELAVIROID:     

A pathogenic non coding RNA that replicates and accumulates in chloroplasts traffics to this organelle through a nuclear-dependent step

Viroids belonging to the family Avsunviroidae are the only functional RNAs known to traffic selectively into chloroplasts. Subcellular targeting is a critical step in guaranteeing their access to the machineries involved in their replication. However, the host mechanisms exploited by these non coding pathogenic RNAs to be selectively imported into chloroplasts are poorly understood. Recently, we provide evidence supporting the idea that the Avsunviroidae have evolved to subvert a signaling mechanism between the nucleus and chloroplasts to regulate their differential compartmentalization into the chloroplast of infected cells. Here, we discuss our model and previous observations that provide biological relevance to our hypothesis.     

Subviral pathogens of plants: viroids and viroidlike satellite RNAs.

Contrary to earlier beliefs, viruses are not the smallest causative agents of infectious diseases. Single-stranded RNAs as small as 246 nucleotides exist in certain higher plants and cause more than a dozen crop diseases. These RNAs have been termed viroids. Despite their extremely limited information content, viroids replicate autonomously in susceptible cells--that is, they do not require helper functions from simultaneously replicating conventional viruses. Viroids are covalently closed circular molecules with a characteristic rodlike secondary structure in which short helical regions are interrupted by internal and bulge loops. Viroids are not translated; they are replicated by a host enzyme (or enzymes) (probably RNA polymerase II) via oligomeric RNA intermediates by a rolling circle mechanism. Viroidlike satellite RNAs resemble viroids in size and molecular structure, but are found within the capsids of specific helper viruses on which they depend for their own replication. These RNAs are of great interest to molecular biology for at least two reasons: 1) they are the smallest and simplest replicating molecules known, and 2) they may represent living fossils of precellular evolution in a hypothetical RNA world.     

Viroids: petite RNA pathogens with distinguished talents

Viroids are small, circular, single-stranded RNA molecules that cause several infectious plant diseases. Viroids do not encode any pathogen-specific peptides but nonetheless, the subviral pathogens replicate autonomously and spread in the plant by recruiting host proteins via functional motifs encoded in their RNA genome. During the past couple of years, considerable progress has been made towards comprehending how viroids interact with their hosts. Here, we summarize recent findings on the structure-function relationships of viroids, their strategies and mechanisms of replication and trafficking, and the identification and characterization of interacting host proteins. We also describe the impact of the RNA silencing machinery of plants on viroid RNAs and how this has started to influence our models of viroid replication and pathogenicity.     

Mature monomeric forms of Hop stunt viroid resist RNA silencing in transgenic plants

Viroids, small non-coding pathogenic RNAs, are able to induce RNA silencing, a phenomenon that has been associated with the pathogenesis and evolution of these small RNAs. It has been recently suggested that viroids may resist this plant defense mechanism. However, the simultaneous degradation of non-replicating full-length viroid RNA, and the resistance of mature forms of viroids to RNA silencing, have not been experimentally demonstrated. Transgenic Nicotiana benthamiana plants expressing a dimeric form of Hop stunt viroid (HSVd) that have the capability to cleave and circularize this viroid RNA were used to address this question. A reporter construct, consisting of a full-length HSVd RNA fused to GFP-mRNA, was agroinfiltrated in these plants and its expression was suppressed. Interestingly, both circular and linear HSVd molecules were stable and able to traffic through grafts in these restrictive conditions, indicating that the mature forms of HSVd are able, in some way, to resist the RNA-silencing mechanism. The observation that a full-length HSVd RNA fused to GFP-mRNA, but not circular and/or linear viroid forms, was fully susceptible to RNA degradation strongly suggests that structures adopted by the free mature monomer protect the pathogenesis-associated forms of the viroid from RNA silencing.     

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.     

Viroids with hammerhead ribozymes: some unique structural and functional aspects with respect to other members of the group.

Viroids, subviral pathogens of plants, are composed of a single-stranded circular RNA of 246-399 nucleotides. Within the 27 viroids sequenced, avocado sunblotch, peach latent mosaic and chrysanthemum chlorotic mottle viroids (ASBVd, PLMVd and CChMVd, respectively) can form hammerhead structures in both of their polarity strands. These ribozymes mediate self-cleavage of the oligomeric RNAs generated in the replication through a rolling circle mechanism, whose two other steps are catalyzed by an RNA polymerase and an RNA ligase. ASBVd, and presumably PLMVd and CChMVd, replicate and accumulate in the chloroplast, whereas typical viroids replicate and accumulate in the nucleus. PLMVd and CChMVd do not adopt a rod-like or quasi rod-like secondary structure as typical viroids do but have a highly branched conformation. A pathogenicity determinant has been mapped in a defined region of the CChMVd molecule.     

Prominent polypurine and polypyrimidine tracts in plant viroids and in RNA of the human hepatitis delta agent.

To seek patterns of nucleotide usage in the three types of circular subviral RNA pathogens, trimer frequencies and nearest-neighbor biases were studied in 12 plant viroid sequences; five sequences of circular plant viral satellite RNAs; and the sequence of RNA from the human hepatitis delta agent. The viroids and RNA of the delta agent contain tracts of polypurines and polypyrimidines which make up substantial portions of their genomes. Such tracts are not common in the virusoids or in the satellite RNA of tobacco ringspot virus. Viroids, the delta hepatitis agent, and the circular satellite RNAs of certain plant viruses have several features in common: all have circular genomic RNA and replicate through an RNA to RNA rolling circle replication cycle. However, virusoids and related satellite RNAs are directly or indirectly dependent on their helper viruses for replication, while the delta agent and viroids are not. The difference in the pattern of nucleotide usage between the plant viral satellite RNAs on the one hand, and viroids and delta RNA on the other, may relate to this difference in replication strategy.     

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.     

Search for Viroids and Other Small RNAs Associated with Forest Damage

We examined whether viroids may be involved as the causative agents of forest damages which have been observed in Germany during the last decade. A crude RNA extract was prepared from healthy and diseased copper beech, spruce, and pine trees and was analysed by two-dimensional gel electrophoresis which resolves RNA bands in the molecular weight range from 25,000 to 10⁶. Viroids are known to consist of single stranded circular RNA and are clearly differentiable in the electrophoresis used here from cellular RNA. Viroids could not be found in any of the samples investigated. In all samples from diseased trees, however, other additional RNA bands were discovered which were more similar to cellular RNA than to viroids in their electrophoretic properties. It is uncertain whether these diseased-associated RNAs are due to an infection or originate from a modified nucleic acid metabolism.