Molecular Plant Pathology Laboratory, USDA/ARS, Room 118 Building 004, 10300 Baltimore Avenue, Beltsville, MD 20705, USA
Correspondence
Robert A. Owens
owensr{at}ba.ars.usda.gov
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the sequences of novel variants described in this study are AY937180AY937194.
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MAIN TEXT |
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In addition to the rod-like native conformation (Fig. 1a), PSTVd can also form a variety of other structures, at least some of which have been shown to have biological relevance [e.g. secondary hairpin II (Qu et al., 1993
) and a tetraloop motif within the central conserved region that is recognized by a host nuclease activity during replication (Baumstark et al., 1997
)]. As discussed by Woese & Pace (1993)
, comparative sequence analysis is the most reliable method to establish higher-order RNA structure; this approach was used 20 years ago to identify five structural/functional domains within PSTVd and related viroids (Keese & Symons, 1985
). Based upon a combination of chemical/enzymic probing and comparative sequence analysis, Gast and colleagues (Gast et al., 1996
; Gast, 2003
) subsequently suggested that one of these domains (the left terminal domain) may have a branched structure. Characterization of the left terminal domain of PSTVd by nuclear magnetic resonance and thermodynamic analysis in vitro, however, has clearly shown the concentration of the branched conformation to be <1 % of that of the rod-like, elongated form (Dingley et al., 2003
). The mutational analysis described below was designed to search for evidence of a branched conformation in vivo.
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As shown in Fig. 1(b), pseudoknot 1 involves complementary GUG and CAC sequences located at PSTVd positions 1416 and 2931. The close proximity of these two sequences allowed them to be mutagenized by using a single pair of degenerate primers, resulting in a mixture of 3072 potentially infectious sequences, each lacking the A residue found at position 30 in wild-type PSTVd_Int. One-third of these transcripts contained the same A/U substitution at position 30 that was noted by Gast (2003)
in his covariation analysis of large pospiviroids; nevertheless, none of the resulting progeny contained a compensatory G/A substitution at position 15. Fifteen of 16 infected plants contained wild-type PSTVd_Int, and the only variant contained a single, spontaneous A/G substitution at position 271 (results not shown).
Similar results were obtained when both sequences forming pseudoknot 2 were mutagenized simultaneously. In this case, covariation at positions 332 and 9, as well as positions 334 and 7, is consistent with a possible tertiary interaction involving positions 330335 (GUUUAG) and 611 (CUAAAC) (Gast, 2003). To reduce the complexity of the mutagenized inoculum, only three positions in each hexanucleotide were mutagenized, resulting in a mixture of 48x48=2304 potentially infectious variants. As shown in Table 1
(see experiment 1), sequence analysis of the resulting progeny revealed that all 10 infected plants contained wild-type PSTVd_Int.
The sequences possibly interacting to form pseudoknot 2 lie far enough from one another that two pairs of degenerate primers were required for mutagenesis. When simultaneous mutagenesis of positions 611 and 330335 failed to yield informative sequence variants, we decided to examine the effect of mutagenizing just one sequence at a time. The resulting bioassay (experiment 2) yielded several informative sequence variants. Mutagenesis of positions 6, 8 and 10 yielded a single novel variant, a sequence in which the C residue at position 6 was replaced by a U. The resulting conversion of a G : C to a G : U base pair should have little effect on the stability of either the rod-like or branched structure of PSTVd, but the weakening of a single G : C pair could have a much greater destabilizing effect on pseudoknot 2, which contains only six base pairs. Mutagenesis of positions 331, 333 and 335 also resulted in several sequence changes (including a spontaneous A/G change at position 334) that could weaken pseudoknot 2.
Having demonstrated the possibility of individually mutating each partner in this potential tertiary interaction, we tried once again to find evidence for sequence covariation in pseudoknot 2. Starting with PSTVd_Int, we first introduced the U/A and G/U changes at positions 333 and 335 that were present in many of the progeny recovered from experiment 2, and then randomized positions 610 as before. Data presented in Table 1 (see experiment 3) revealed that 15 of 20 inoculated plants became infected none with wild-type PSTVd. Each variant retained the U/A and G/U changes at positions 333 and 335 that were present in the inoculum; furthermore, changes consistent with the potential interaction of positions 330335 and 611 were also detected at positions 6 and 8.
In order to completely restore WatsonCrick pairing with the modified GUUAAU sequence at positions 330335, new variants would be expected to contain an A/U substitution at position 8 and either a C/A or a C/G substitution at position 6. Both of these variants were recovered from single infected plants, but neither was the predominant sequence in the population. Note, however, that the variants recovered from a majority of the plants examined (eight of 15) contained a C/U substitution at position 8. One effect of this change is to recreate a pseudoknot 2 containing five rather than six contiguous base pairs. At best, the observed pattern of changes was only partially consistent with pseudoknot 2 formation.
The appearance of spontaneous sequence changes during passage of viroid mutants in vivo often signals a significant loss of fitness by the initial mutant (Góra-Sochacka et al., 1997). Although many such spontaneous changes appear to be neutral in nature (Matou
ek et al., 2001
), others restore important interactions disrupted by the initial mutagenesis [e.g. secondary hairpin II (Qu et al., 1993
)]. Thus, if formation of pseudoknot 2 was essential for PSTVd replication, one would expect at least some spontaneous changes to restore base pairing between nt 611 and 330335. In experiment 4, groups of five Rutgers tomato seedlings were inoculated with PSTVd variants recovered from our initial mutant screens and then monitored for changes in symptom expression consistent with an increase in viroid fitness. Fig. 2
contrasts the appearance of representative plants from this bioassay and sequence changes detected in the progeny are summarized in Table 1
(see experiment 4).
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Although the Subviral RNA Database (http://subviral.med.uottawa.ca) now contains the sequences of nearly 100 different PSTVd variants, virtually all variability is located outside portions of the left terminal domain that are potentially involved in pseudoknot formation. Thus, the covariation analysis of Gast (2003) was based entirely on comparisons of PSTVd with related pospiviroid species. Our mutational analysis represents the first attempt to obtain direct experimental evidence for pseudoknot formation in PSTVd. As described above, we were able to generate sequence diversity affecting only pseudoknot 2, a putative WatsonCrick pairing between PSTVd positions 611 (CUAAAC) and 330335 (GUUUAG). Pseudoknot 2 is the least conserved of the two potential pseudoknots and comparable interactions in the other large pospiviroids involve as few as three and as many as nine base pairs (Gast, 2003
).
Structural calculations (Lück et al., 1999) suggest that several of our mutagenesis-induced sequence changes could facilitate formation of pseudoknot 2 indirectly by destabilizing the rod-like structure of the left terminal domain (results not shown). Only two variants, however, contained all of the compensatory changes necessary to maintain WatsonCrick base pairing completely in pseudoknot 2. We note that these compensatory changes did not appear spontaneously during passage of the initial mutant, but required a second round of in vitro mutagenesis. Overall, the pattern of mutagenesis-induced and spontaneous sequence changes observed provides little support for the existence of pseudoknot 2.
Pseudoknot 1, a second, more highly conserved potential interaction between PSTVd positions 1416 and 2931, did not respond to mutagenesis. Several explanations for such a result are possible, but the failure to recover even a single variant is surprising. Several large pospiviroids, including the closely related Citrus exocortis viroid and Tomato apical stunt viroid, contain a central U : A rather than an A : U base pair, and the reappearance of wild-type PSTVd_Int in 15 of 16 infected plants indicates that there was ample opportunity for sequence evolution/selection in vivo. A recent report (Wang et al., 2004) suggests that RNA silencing exerts considerable pressure on viroids and viral satellites to maintain a compact, highly base-paired structure. Our results, together with those from in vitro structural studies with model RNAs (Dingley et al., 2003
), suggest strongly that the left terminal domain of PSTVd does not assume a branched or folded conformation containing pseudoknots in vivo.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Dingley, A. J., Steger, G., Esters, B., Riesner, D. & Grzesiek, S. (2003). Structural characterization of the 69 nucleotide potato spindle tuber viroid left-terminal domain by NMR and thermodynamic analysis. J Mol Biol 334, 751767.[CrossRef][Medline]
Gast, F.-U. (2003). A new structural motif in the left terminal domain of large viroids identified by covariation analysis. Virus Genes 26, 1923.[CrossRef][Medline]
Gast, F.-U., Kempe, D., Spieker, R. L. & Sänger, H. L. (1996). Secondary structure probing of potato spindle tuber viroid (PSTVd) and sequence comparison with other small pathogenic RNA replicons provides evidence for central non-canonical base-pairs, large A-rich loops, and a terminal branch. J Mol Biol 262, 652670.[CrossRef][Medline]
Góra-Sochacka, A., Kierzek, A., Candresse, T. & Zagórski, W. (1997). The genetic stability of potato spindle tuber viroid (PSTVd) molecular variants. RNA 3, 6874.
Hadidi, A., Flores, R., Randles, J. W. & Semancik, J. S. (editors) (2003). Viroids. Collingwood, Australia: CSIRO Publishing.
Keese, P. & Symons, R. H. (1985). Domains in viroids: evidence of intermolecular RNA rearrangements and their contribution to viroid evolution. Proc Natl Acad Sci U S A 82, 45824586.
Lück, R., Gräf, S. & Steger, G. (1999). ConStruct: a tool for thermodynamic controlled prediction of conserved secondary structure. Nucleic Acids Res 27, 42084217.
Matouek, J., Patzak, J., Orctová, L., Schubert, J., Vrba, L., Steger, G. & Riesner, D. (2001). The variability of hop latent viroid as induced upon heat treatment. Virology 287, 349358.[CrossRef][Medline]
Owens, R. A., Thompson, S. M. & Kramer, M. (2003). Identification of neutral mutants surrounding two naturally occurring variants of Potato spindle tuber viroid. J Gen Virol 84, 751756.
Podleckis, E. V., Hammond, R. W., Hurtt, S. W. & Hadidi, A. (1993). Chemiluminescent detection of potato and pome fruit viroids by digoxygenin-labeled dot blot and tissue blot hybridization. J Virol Methods 43, 147158.[CrossRef][Medline]
Qu, F., Heinrich, C., Loss, P., Steger, G., Tien, P. & Riesner, D. (1993). Multiple pathways of reversion in viroids for conservation of structural elements. EMBO J 12, 21292139.[Abstract]
Stasys, R. A., Dry, I. B. & Rezaian, M. A. (1995). The termini of a new citrus viroid contain duplications of the central conserved regions from two viroid groups. FEBS Lett 358, 182184.[CrossRef][Medline]
Steger, G. & Riesner, D. (2003). Molecular characteristics. In Viroids, pp. 1529. Edited by A. Hadidi, R. Flores, J. W. Randles & J. S. Semancik. Collingwood, Australia: CSIRO Publishing.
Wang, M.-B., Bian, X.-Y., Wu, L.-M. & 10 other authors (2004). On the role of RNA silencing in the pathogenicity and evolution of viroids and viral satellites. Proc Natl Acad Sci U S A 101, 32753280.
Woese, C. R. & Pace, N. R. (1993). Probing RNA structure, function, and history by comparative analysis. In The RNA World, pp. 91117. Edited by R. F. Gesteland & J. F. Atkins. Plainview, NY: Cold Spring Harbor Press.
Received 30 December 2004;
accepted 23 February 2005.
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