Eggplant Latent Viroid , the Candidate Type Species for a New Genus within the Family Avsunviroidae (Hammerhead Viroids)

Abstract

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.Viroids and satellite RNAs have been implicated in several plant diseases. Whereas viroids replicate autonomously (12), satellite RNAs depend functionally on helper viruses and are encapsidated by their coat proteins (17). A survey in Eastern Spain for detecting small infectious RNAs in vegetables identified a viroid-like RNA in eggplant (Solanum melongena L. cv. Sonja) (8). Denaturing polyacrylamide gel electrophoresis (PAGE) revealed two bands with the mobilities expected for the circular and linear forms, and treatment with RNase, but not with DNase, caused their disappearance. Inoculation of eggplant seedlings with preparations containing the viroid-like RNA showed the presence of the same RNA in some of the inoculated plants, which remained symptomless, and its absence in the noninoculated controls. On this basis, the RNA was named Eggplant latent viroid (ELVd), although the possibility that it could be a viroid-like satellite RNA could not be dismissed (8). Attempts to transmit ELVd to tomato, chrysanthemum, cucumber, and citron, which support the replication of different viroids, failed, suggesting a restricted host range, and riboprobes specific for Citrus exocortis viroid (CEVd), Hop stunt viroid, and Apple scar skin viroid did not hybridize with ELVd, suggesting that it was not a member of the genus Pospiviroid, Hostuviroid, or Apscaviroid (8). These genera belong to the family Pospiviroidae, whose members have a central conserved region and lack hammerhead ribozymes (12). These data, together with the low stability of ELVd circular forms in vitro (9), were indicative of a peculiar structure and prompted us to study the biological and molecular properties of ELVd.Biological properties of ELVd. Eggplant seedlings (cv. Redonda morada) inoculated with ELVd and kept in a greenhouse were used as the source of ELVd. To confirm that ELVd could replicate autonomous...

Homology-independent discovery of replicating pathogenic circular RNAs by deep sequencing and a new computational algorithm

“…In fact, the circular RNA species identified in this study may be such a satellite RNA, which would explain why it alone was not infectious. In addition, the pattern of sequence variation among the nonconserved nucleotides in the GHVd-like RNA hammerhead ribozymes shows features associated with hammerhead ribozymes encoded by either viroids or circular satellite RNAs (27,35,36).…”

Arabidopsis thaliana has the enzymatic machinery for replicating representative viroid species of the family Pospiviroidae

“…Twenty-six viroid species, which contain in their RNAs a central conserved region, belong to the family Pospiviroidae, whereas four viroid species that lack a central conserved region but are able to self-cleave through hammerhead ribozymes are grouped in the family Avsunviroidae (12,19). Viroid replication occurs through an RNA-based rolling-circle mechanism (20) in which the infecting monomeric (ϩ) circular RNA is transcribed by an RNA polymerase into head-to-tail (Ϫ) multimers that serve as templates for a second RNA-RNA transcription step.…”

The predominant circular form of avocado sunblotch viroid accumulates in planta as a free RNA adopting a rod-shaped secondary structure unprotected by tightly bound host proteins

“…Although the ml (+) and (À) RNAs of eggplant latent viroid (ELVd, 333 nt) [35] are clearly discriminated by electrophoresis in gels of this porosity [36], they might not have sufficient power to resolve RNAs of smaller size like those of ASBVd. Therefore, we performed our experiments in 8 % gels in which, according to earlier data, ml ASBVd (+) and (À) RNAs migrate differentially [23].…”

“…Considering that conventional SHAPE in vivo demands relatively high viroid titres in infected tissues, we discarded PLMVd [44] and CChMVd [45] due to their poor accumulation in planta. Between the remaining two members of Avsunviroidae, the eggplant latent viroid (ELVd) [35] and ASBVd [11], we chose the latter because despite its natural host being a woody plant, the isolate of this viroid, which we have been working with over the years (GenBank accession number J02020 with the substitution C213fiU), accumulates in vivo to levels comparable to the 5S ribosomal RNA [12,21], much higher than those reached by ELVd in eggplant. There are modifications more sensitive than conventional SHAPE [40,46], but some entail additional steps, particularly single-stranded adapter ligation, which limit the reliability of the results.…”