Avocado Sunblotch Viroid Research Articles

A chloroplast protein binds a viroid RNA in vivo and facilitates its hammerhead-mediated self-cleavage.

Viroids, small single-stranded circular RNAs (246-401 nucleotides), do not have mRNA capacity and must recruit host proteins to assist in the steps of their biological cycle. The nature of these cellular factors is poorly understood due to a lack of reliable experimental approaches. Here, to screen for host proteins interacting with viroid RNAs in vivo, we UV-irradiated avocado leaves infected with avocado sunblotch viroid (ASBVd), the type member of chloroplast viroids containing hammerhead ribozymes. This resulted in the detection of several ASBVd-host protein adducts. Tandem mass spectrometry analysis of the most abundant cross-linked species identified the protein component as two closely related chloroplast RNA-binding proteins (PARBP33 and PARBP35) of a family whose members previously have been shown to be involved in stabilization, maturation and editing of chloroplast transcripts. PARBP33 behaves as an RNA chaperone that stimulates in vitro the hammerhead-mediated self-cleavage of the multimeric ASBVd transcripts that result from rolling circle replication, indicating that this reaction, despite its RNA-based mechanism, is facilitated by proteins. The structural and functional parallelism between PARBP33 and PARBP35, and some proteins involved in viral RNA replication, indicates that viroids and RNA viruses recruit similar host proteins for their replication.

Transcription of potato spindle tuber viroid by RNA polymerase II starts predominantly at two specific sites.

Pospiviroidae, with their main representative potato spindle tuber viroid (PSTVd), are replicated via a rolling circle mechanism by the host-encoded DNA-dependent RNA polymerase II (pol II). In the first step, the (+)-strand circular viroid is transcribed into a (-)-strand oligomer intermediate. As yet it is not known whether transcription is initiated by promotors at specific start sites or is distributed non-specifically over the whole circle. An in vitro transcription extract was prepared from a non-infected potato cell culture which exhibited transcriptional activity using added circular PSTVd (+)-strand RNA as template. In accordance with pol II activity, transcription could be inhibited by alpha-amanitin. RT-PCR revealed the existence of at least two different start sites and primer extension identified these as nucleotides A(111) and A(325). The sequences of the first 7 nt transcribed are very similar, (105)GGAGCGA(111) and (319)GGGGCGA(325). GC-boxes are located at a distance of 15 and 16 nt upstream, respectively, in the native viroid structure, which may act to facilitate initiation. The GC-boxes may have a similar function to the GC-rich hairpin II in the (-)-strand intermediate, as described previously. The results are compared with the corresponding features of avocado sunblotch viroid, which belongs to a different family of viroids and exhibits different transcription initiation properties.

Sequence Diversity among Avocado Sunblotch Viroids Isolated from Single Avocado Trees

In previous studies we developed an avocado sunblotch viroid (ASBVd) specific reverse transcription-polymerase chain reaction (RT-PCR) protocol and tested all avocado germplasm accessions in the U.S. National Germplasm Repository (NGR) for ASBVd. It was found that 19% of the accessions were infected with the viroid. To characterize the population of sequence variants, single trees of seven accessions with a history of infection were selected. RT-PCR products were cloned and sequenced from these trees and a total of 60 sequence variants were identified from 122 clones. Most of the variants were found to be unique to the individual tree from which they were isolated. The fraction of unique variants was 81% in ‘Lima Late’, 81% in ‘Aycock Red’, 67% in ‘Hawaii’, and 60% in ‘Young Special’. Analysis of the sequence data suggests that variants evolved within a tree rather than arising from multiple infection events, supporting the quasispecies concept for ASBVd. Changes in the right terminal loop (Rtl), previously associated with leaf variegation and bleaching, were found in all variants isolated from bleached tissue; however, 16 clones isolated in non-symptomatic tissue also had changes in the Rtl. The addition of a U between bases 115 and 118 was found in 21 of the 23 clones isolated from the bleached tissue but not found in the 99 clones isolated from green tissue. These data suggest that the insertion of a U between bases 115 and 118 is responsible for the bleaching condition.

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 α-amanitin had no effect on gene or on viroid transcription, but tagetitoxin (5–10 μM) prevented transcription of all these genes without affecting synthesis of ASBVd strands; only at higher tagetitoxin concentrations (50–100 μM) 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.

Avsunviroidae family: Viroids containing hammerhead ribozymes

This chapter focuses on the second viroid family, whose members are also referred to as hammerhead viroids, taking into account their most outstanding feature. If the word “small” is the first to come to mind when considering viroids, perhaps the second word is “hammerhead,” because this class of ribozymes, which because of its structural simplicity has an enormous biotechnological potential, is described in avocado sunblotch viroid (ASBVd) as well as in a viroid-like satellite RNA. The most outstanding feature of the Avsunviroidae members is their potential to adopt hammerhead structures in both polarity strands and to self-cleave in vitro accordingly. Viroids differ from viruses not only in their genome size but also in other fundamental aspects, prominent among which is the lack of messenger activity of both viroid RNAs and their complementary strands.

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.

Chrysanthemum chlorotic mottle viroid: unusual structural properties of a subgroup of self-cleaving viroids with hammerhead ribozymes.

The causal agent of chrysanthemum chlorotic mottle (CChM) disease has been identified, cloned, and sequenced. It is a viroid RNA (CChMVd) of 398-399 nucleotides. In vitro transcripts with the complete CChMVd sequence were infectious and induced the typical symptoms of the CChM disease. CChMVd can form hammerhead structures in both polarity strands. Plus and minus monomeric CChMVd RNAs self-cleaved during in vitro transcription and after purification as predicted by these structures, which are stable and most probably act as single hammerhead structures as in peach latent mosaic viroid (PLMVd), but not in avocado sunblotch viroid (ASBVd). Moreover, the plus CChMVd hammerhead structure also appears to be active in vivo, because the 5' terminus of the linear plus CChMVd RNA isolated from infected tissue is that predicted by the corresponding hammerhead ribozyme. Both hammerhead structures of CChMVd display some peculiarities: the plus self-cleaving domain has an unpaired A after the conserved A9 residue, and the minus one has an unusually long helix II. The most stable secondary structure predicted for CChMVd is a branched conformation that does not fulfill the rod-like or quasi-rod-like model proposed for the in vitro structure of most viroids with the exception of PLMVd, whose proposed secondary structure of lowest free energy also is branched. The unusual conformation of CChMVd and PLMVd is supported by their insolubility in 2 M LiCl, in contrast to ASBVd and a series of representative non-self-cleaving viroids that are soluble under the same high salt conditions. These results support the classification of self-cleaving viroids into two subgroups, one formed by ASBVd and the other one by PLMVd and CChMVd.

Application of RT-PCR for Indexing Avocado Sunblotch Viroid.

A method for the routine detection of avocado sunblotch viroid (ASBVd) in nucleic acid extracts of infected avocado tissues by reverse transcription-polymerase chain reaction (RT-PCR) was developed using ASBVd-specific primers. Amplified cDNA products were analyzed by electrophoresis on nondenaturing 6% polyacrylamide slab gels. The size of the major RT-PCR product from ASBVd-infected tissue was estimated to be 250 bp. This product was absent from amplified extracts of uninfected tissue. The amplification product from ASBVd was sequenced by the dideoxynucleotide chain termination method, and the sequence was over 97% identical to the published sequence. The RT-PCR assay is sensitive enough to allow viroid detection without requiring large amounts of tissue, highly purified ASBVd, or molecular hybridization.

The RNA of both polarities of the peach latent mosaic viroid self-cleaves in vitro solely by single hammerhead structures.

Hammerhead self-cleavage of dimeric, monomeric, truncated and mutated transcripts derived from both polarities of the peach latent mosaic viroid (PLMVd) were characterized. In contrast to some results previously published for a very close sequence variant (see ref. 1), these RNAs exhibit a virtually identical self-cleavage during transcription and after purification. By self-cleavage of dimeric transcripts with normal and mutated hammerhead domains and by complementation experiments, we show that the cleavage reactions involve only single hammerhead structures. This observation contrasts with the case of avocado sunblotch viroid (ASBVd), the other self-cleaving viroid, whose mechanism involves mostly double hammerhead structures, whereas single hammerhead cleavage is associated with viroid-like plant satellite RNAs. The difference in stability between the native secondary structures adopted by viroids and the autocatalytic structures, including the hammerhead motif, governs the efficiency of the self-cleavage reaction. The transition between these conformers is the limiting step in catalysis and is related exclusively to the left arm region of PLMVd secondary structure, which includes the hammerhead sequences. Most of the mutations between the variant we used and the sequence variant previously published are located in this left arm region, which may explain to a great extent the differences in their cleavage efficiency. No interactions with long-range sequences contributing to the autocatalytic tertiary structure were revealed in these experiments.

Detection of Potato Spindle Tuber Viroid in Avocado Growing in Peru

Potato spindle tuber viroid (PSTVd) was detected in avocado trees growing at several locations in Peru by using nucleic acid spot hybridization assays. A viroidlike nucleic acid with a mobility similar to that of PSTVd was also found by return polyacrylamide gel electrophoresis analysis of RNA extracted from several trees, and a mild strain of PSTVd was recovered from Nicandra physalodes inoculated with leaf sap from affected avocados. Sequence analysis of cDNAs obtained using the polymerase chain reaction with primers to PSTVd indicated the presence of three different sequence variants of PSTVd in the avocado trees. The PSTVd infections were often latent, but some trees coinfected with avocado sunblotch viroid showed symptoms that included bunchiness of the inflorescence, decrease in both fruit size and number, and eventual decline and death. This is the first report of the isolation of PSTVd from a natural host other than potato

Replication of avocado sunblotch viroid: evidence for a symmetric pathway with two rolling circles and hammerhead ribozyme processing.

The structure of a series of RNAs extracted from avocado infected by the 247-nt avocado sunblotch viroid (ASBVd) was investigated. The identification of multistranded complexes containing circular ASBVd RNAs of (+) and (-) polarity suggests that replication of ASBVd proceeds through a symmetric pathway with two rolling circles where these two circular RNAs are the templates. This is in contrast to the replication of potato spindle tuber viroid and probably of most of its related viroids, which proceeds via an asymmetric pathway where circular (+)-strand and linear multimeric (-)-strand RNAs are the two templates. Linear (+) and (-) ASBVd RNAs of subgenomic length (137 nt and about 148 nt, respectively) and one linear (+)-strand ASBVd RNA of supragenomic length (383-384 nt) were also found in viroid-infected tissue. The two linear (+)-strand RNAs have the same 5'- and 3'-terminal sequences, with the supragenomic species being a fusion product of the monomeric and subgenomic (+)-strand ASBVd RNAs. The 3' termini of these two (+)-strand molecules, which at least in the subgenomic RNA has an extra nontemplate cytidylate residue, could represent sites of either premature termination of the (+)-strands or specific initiation of the (-)-strands. The 5' termini of sub- and supragenomic (+)-strand and the 5' terminus of the subgenomic (-)-strand ASBVd RNA are identical to those produced in the in vitro self-cleavage reactions of (+) and (-) dimeric ASBVd RNAs, respectively. These observations strongly suggest that the hammerhead structures which mediate the in vitro self-cleavage reactions are also operative in vivo.

Detection of avocado sunblotch viroid in chloroplasts of avocado leaves by in situ hybridization.

In situ hybridization experiments were carried out to detect avocado sunblotch viroid (ASBVd) in foliar tissue of avocado, using a digoxigenin-labelled RNA probe complementary to the ASBVd-RNA in sections of aldehyde-fixed, LRGold-embedded leaf samples. Detection of the probe was made through anti-digoxigenin antibody and protein-A colloidal gold (20 nm). Seventy to 80% of the signals came from chloroplast while the cytoplasm and vacuole were labelled with ca. 10% of the gold particles. This is in contrast with the subcellular localization of potato spindle tuber viroid and some other related viroids, which are mainly found in the nucleus.

In situ hybridization localizes avocado sunblotch viroid on chloroplast thylakoid membranes and coconut cadang cadang viroid in the nucleus

SummaryViroids, small single‐stranded circular RNA molecules, are the smallest known infectious agents in Nature. The apparent inability of viroids to encode for proteins means that they must rely fully on host functions for their replication. The specific ultrastructural localization of viroids is fundamental to the determination of their replication strategies. In this paper the first in situ hybridization study to localize viroids within the cell at the electron microscope level is reported. Biotin‐labelled RNA probes were used with subsequent detection by gold‐labelled monoclonal anti‐biotin antibodies to localize avocado sunblotch viroid and coconut cadang cadang viroid. Avocado sunblotch viroid was located in chloroplasts, mostly on the thylakoid membranes of cells from infected leaves of avocado (Persea americana). In contrast, coconut cadang cadang viroid was located in the nucleolus and nucleoplasm of cells of infected leaves of oil palm (Elaeis guineensis), with a higher concentration in the nucleolus. The results provide insight on the potential host RNA polymerases involved in the replication of these two viroids.

Avocado sunblotch disease: a persistent viroid infection in which variants are associated with differential symptoms.

Variants of avocado sunblotch viroid (ASBVd) of between 247 and 250 nucleotides in length have been recovered from diseased avocado tissues. The sunblotch syndrome covers a complex pattern of disease symptoms which are associated with infection by variants of ASBVd. The viroid species are designated ASBVd-B, ASBVd-V and ASBVd-Sc from their association with bleached, variegated or symptomless carrier tissues respectively. Host-viroid interactions and structural relationships among the variants suggest a transition in sunblotch disease from a severe acute to a persistent mild form of infection.

The 5' end Generated in the in vitro Self-Cleavage Reaction of Avocado Sunblotch Viroid RNAs is Present in Naturally Occurring Linear Viroid Molecules

The 5' termini of linear forms of avocado sunblotch viroid (ASBVd) isolated from infected tissue have been determined by primer extension analyses. According to the different termini identified, four main populations of linear viroid molecules were found. One of them had the same 5' end as the one produced in the in vitro self-cleavage reaction of ASBVd dimeric transcripts mediated by a hammerhead structure, suggesting a possible involvement of this in vitro reaction in the in vivo replication mechanism of ASBVd.

Characterization of RNAs specific to avocado sunblotch viroid synthesized in Vitro by a cell-free system from infected avocado leaves

Analysis by molecular hybridization of the RNAs transcribed by a cell-free fraction from avocado infected with avocado sunblotch viroid (ASBV) demonstrated the presence of newly synthesized viroid-specific sequences, most of which were of the same polarity as the mature infectious viroid RNA. Treatment of the cell-free fraction with DNase reduced the total synthesis of RNA considerably, but it did not influence that of the ASBV-specific RNAs, indicating that the latter were transcribed on an RNA template. Inhibition studies with α-amanitin showed that the synthesis of ASBV-specific RNAs was not affected by concentrations of 1 and 200,μg/ml of the drug, which typically inhibit RNA polymerase I I and III, respectively, from most animal and plant systems. These results suggest that either RNA polymerase I or an unidentified RNA polymerase activity resistant to α-amanitin, acting on an RNA template, plays a role in the replication of ASBV, whereas for the rest of the viroids studied so far it appears that RNA polymerase II is involved. Analysis by polyacrylamide gel electrophoresis under partially and fully denaturing conditions of the ASBV-specific RNAs synthesized in vitro showed that they contain unit and longer than unit length viroid strands, probably associated in complexes with single- and double-stranded regions. The structural properties of these complexes are similar to those of the RNAs accumulating in vivo in viroid-infected tissues, which are the postulated replicative intermediates of the rolling-circle mechanism proposed for viroid synthesis.

Alternative hammerhead structures in the self-cleavage of avocado sunblotch viroid RNAs

The plus and minus RNAs of the 247 nt avocado sunblotch viroid (ASBV) undergo site specific RNA self-cleavage reactions in vitro. As with several other self-cleaving RNAs, we proposed hammerhead secondary structures for the sequence around the site of self-cleavage of both RNAs. We have shown previously that, during transcription of a dimeric plus ASBV RNA, a double-hammerhead structure formed and was necessary for self-cleavage. Here, we show that the purified full-length dimeric plus RNA, when incubated under our standard self-cleavage conditions, also self-cleaved by a double-hammerhead structure. In contrast, a dimeric minus ASBV RNA self-cleaved by a double-hammerhead structure during transcription, but by a single-hammerhead structure after purification. This illustrates the importance of the pathway of folding of the RNA in determining which active self-cleaving structure is formed.

Subcellular location of avocado sunblotch viroid in avocado leaves

The subcellular accumulation site of avocado sunblotch viroid (ASBV) in avocado leaves has been investigated. By differential centrifugation techniques, ASBV was predominantly found in a form non-sedimentable at 100 000 x g for 2 h. A minor proportion of the viroid was also detected in association with larger cellular constituents. This association appeared to be mostly artefactual, since a very similar distribution pattern of ASBV was observed in control experiments with non-infected leaves processed with grinding medium containing purified viroid. Moreover, the ASBV contents of crude nuclear and chloroplastic preparations were considerably reduced after their purification, and small amounts of viroid still bound to the purified organella were also observed in controls with non-infected tissue and externally added ASBV. In all these experiments the distribution of the viroid paralleled that of the cytoplasmic 4S RNAs. Therefore, ASBV appears to have the same subcellular location as coconut cadang-cadang viroid, being the two only exceptions to the so far existing generalization pointing to the nucleus as the major accumulation site for viroids.