Detection of avocado sunblotch viroid variants using fluorescent single-strand conformation polymorphism analysis.

A specific reverse transcription-polymerase chain reaction (RT-PCR) protocol has been developed for routine detection of avocado sunblotch viroid (ASBVd). Modifications in this diagnostic technique were made to enable fluorescent detection and variant identification using automated capillary electrophoresis (CE) and fluorescent single-strand conformation polymorphism (SSCP) analysis. Sixteen sequence variants characterized in a previous study were analyzed using CE-SSCP on two ABI 310 Genetic Analyzers. Significant differences were detected between data obtained from the two ABI 310 Genetic Analyzers indicating that an internal control must be run concurrently with the samples. The 16 variants could be classified into 11 groups based on the SSCP patterns. The statistical analysis of the migration rate data provided support for the visual differences in SSCP patterns. The use of SSCP in the ASBVd assay is easily accomplished and gives an estimate of the number of variants in crude samples extracted from infected avocado plants.

Characterization of the initiation sites of both polarity strands of a viroid RNA reveals a motif conserved in sequence and structure.

Viroids replicate through a rolling-circle mechanism in which the infecting circular RNA and its complementary (-) strand are transcribed. The precise site at which transcription starts was investigated for the avocado sunblotch viroid (ASBVd), the type species of the family of viroids with hammerhead ribozymes. Linear ASBVd (+) and (-) RNAs begin with a UAAAA sequence that maps to similar A+U-rich terminal loops in their predicted quasi-rod-like secondary structures. The sequences around the initiation sites of ASBVd, which replicates and accumulates in the chloroplast, are similar to the promoters of a nuclear-encoded chloroplastic RNA polymerase (NEP), supporting the involvement of an NEP-like activity in ASBVd replication. Since RNA folding appears to be kinetically determined, the specific location of both ASBVd initiation sites provides a mechanistic insight into how the nascent ASBVd strands may fold in vivo. The approach used here, in vitro capping and RNase protection assays, may be useful for investigating the initiation sites of other small circular RNA replicons.

A chloroplastic RNA polymerase resistant to tagetitoxin is involved in replication of avocado sunblotch viroid.

Avocado sunblotch viroid (ASBVd), the type species of the family Avsunviroidae, replicates and accumulates in the chloroplast. Two main chloroplastic RNA polymerases have been described: the plastid-encoded polymerase (PEP) with a multisubunit structure similar to the Escherichia coli enzyme and a single-unit nuclear-encoded polymerase (NEP) resembling phage RNA polymerases. On a different basis, sensitivity to tagetitoxin, two major RNA polymerase activities, tagetitoxin sensitive (TS) and resistant (TR), have been found in plastids. The most plausible candidates for the TS and TR RNA polymerases are PEP and NEP, respectively. To gain an insight into the enzymology of the polymerization of ASBVd strands, purified chloroplast preparations from ASBVd-infected leaves were assayed for their in vitro ability to transcribe ASBVd RNAs together with some representative genes (psbA, 16SrDNA, accD, and rpoB) of the three classes of chloroplastic genes according to their promoter structure. High concentrations of alpha-amanitin had no effect on gene or on viroid transcription, but tagetitoxin (5-10 microM) prevented transcription of all these genes without affecting synthesis of ASBVd strands; only at higher tagetitoxin concentrations (50-100 microM) was a 25% inhibition observed. These results suggest that NEP is the RNA polymerase required in ASBVd replication, although the participation of another TR RNA polymerase from the chloroplast cannot be excluded.

Complexes containing both polarity strands of avocado sunblotch viroid: identification in chloroplasts and characterization.

RNA analysis by nondenaturing polyacrylamide gel electrophoresis and Northern blot hybridization of avocado chloroplasts purified from protoplasts of leaves infected by avocado sunblotch viroid (ASBVd) revealed the main ASBVd-specific bands found previously in preparations of total leaf RNA: the monomeric, dimeric and subgenomic RNAs, and two bands, x and y. After RNase treatment in high ionic strength, bands x and y remained resistant, indicating a high content of double-stranded RNAs, whereas the other viroid-specific and cellular RNAs were degraded. Analysis by denaturing polyacrylamide gel electrophoresis and Northern blot hybridization showed that the major constituents of the purified y and x bands were the monomeric circular and linear ASBVd forms of both polarities, but band y contained additionally multimeric ASBVd RNAs, also of both polarities, that probably cause its slower migration in nondenaturing gels. After RNase treatment, the composition of band y was essentially unaffected, but only the monomeric linear ASBVd RNAs of both polarities was recovered from band x. However, in the presence of higher RNase concentrations, band y was converted into band x, indicating that they are closely related. The structure of complexes x and y, containing minus ASBVd strands and particularly the monomeric circular form, supports a role of replicative intermediates in the symmetric rolling circle mechanism proposed for ASBVd, whereas their localization in the chloroplast is strong evidence in favor of this organelle as the replication site of ASBVd.