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Short Communication |
1 Plant Pathology Laboratory, Faculty of Agriculture and Life Sciences, Hirosaki University, Hirosaki 036-8561, Japan
2 Molecular Plant Pathology Laboratory, Plant Sciences Institute, Beltsville Agricultural Research Center (USDA/ARS), Beltsville, MD 20705, USA
3 Gene Research Center, Hirosaki University, Hirosaki 036-8561, Japan
Correspondence
Teruo Sano
sano{at}cc.hirosaki-u.ac.jp
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ABSTRACT |
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Supplementary material is available with the online version of this paper.
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MAIN TEXT |
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). Unlike RNA viruses, the genetic information encoded by PSTVd and other viroids is not translated into protein, and viroid replication is completely dependent on the host's transcriptional machinery (Flores et al., 2005; Ding & Itaya, 2007).
As might be expected from their highly base-paired structure and RNA–RNA mode of replication, viroids have been shown to induce RNA silencing. Accumulation of viroid-specific small RNA (Vd-sRNA) was first detected in tomato plants infected with PSTVd (Itaya et al., 2001; Papaefthimiou et al., 2001). Later, three viroids belonging to the family Avsunviroidae, that replicate in the chloroplast rather than the nucleus, were also reported to induce RNA silencing (Martínez de Alba et al., 2002; Markarian et al., 2004). Vd-sRNA appears to play an important role in mediating viroid symptom expression (Markarian et al., 2004; Wang et al., 2004).
Previous studies from our laboratory have suggested that RNA silencing could be responsible for the recovery phenomenon observed in PSTVd-infected tomato during the later stages of infection (Sano & Matsuura, 2004). To examine the relationship between RNA silencing and recovery in greater detail, we have carried out time-course analyses of Vd-sRNA accumulation using different viroid–host combinations. In the course of these analyses, we observed the presence of two size classes of Vd-sRNAs in plants. Nucleotide sequence analysis revealed the presence of a previously undescribed group of small RNAs derived from negative-strand PSTVd RNA, thereby indicating that the process by which these Vd-sRNAs are generated may be more complicated than previously believed.
In the first experiment, 42 tomato (Solanum lycopersicum, cv. Rutgers) seedlings were inoculated with PSTVd-Intermediate strain at the cotyledon stage. Between 2 and 45 days post-inoculation (d.p.i.), leaves periodically collected from corresponding positions (the first true leaf to the apical meristem) on three plants chosen at random were combined, and the pooled samples were processed for the analysis of PSTVd progeny and PSTVd-specific small RNAs (denoted as srPSTVd following Itaya et al., 2007). Data presented in Fig. 1 compare the accumulation of PSTVd progeny and srPSTVds at later times, i.e. between 14 and 45 d.p.i.
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revealed the presence of detectable concentrations of srPSTVd in leaves 1–5. At 16 d.p.i., as symptoms of epinasty and stunting began to intensify, data shown in Fig. 1(b) revealed that PSTVd accumulation also increased, reaching maximum levels in leaves 3–10 on day 18 and maintaining these levels until sometime between days 30 and 45. As leaves 9–10 reached full expansion between 30 and 45 d.p.i., symptom severity decreased dramatically.
As shown in Fig. 1(a), older, fully expanded leaves contained two distinct size classes of srPSTVd, i.e. molecules containing 21–22 nt and ca 24 nt. This is most clearly seen for leaves 2–4 (14 d.p.i.), leaves 2–6 (16 d.p.i.), leaves 1–7 (18 d.p.i.) and leaves 3–10 (25 d.p.i.). In contrast, only shorter srPSTVd (ca 21 nt) were visible in the upper expanding leaves of PSTVd-infected tomato, i.e. leaf 5 (14 d.p.i.), leaf 7 (16 d.p.i.), leaves 8–9 and the flower bud (18 d.p.i.), and leaf 10 (25 d.p.i.). The result indicates that at 14–18 d.p.i. just after the induction of RNA silencing, only short srPSTVd was detectable in the leaf primordia near the shoot apex; in contrast, both short and long molecules were clearly detectable in the fully expanded, lower leaves showing prominent disease symptoms. When the Northern analysis data were replotted in a schematic, semiquantitative fashion (see Fig. 1c), accumulation of longer srPSTVd could be seen to consistently lag 2–3 days behind that of the shorter species (Fig. 1c). Late in infection (i.e. 30–45 d.p.i.), srPSTVd became more heterogeneous in size.
To get a clearer understanding of srPSTVd accumulation in the uppermost leaves and shoot apex, a second experiment was performed (Fig. 2a). Pooled samples from leaves 1–9 and the apical meristem were assembled from 50 plants at 15 d.p.i. and analysed for the presence of srPSTVds. Consistent with the pattern seen in the first experiment (see Fig. 1a; 14 and 16 d.p.i.), leaves 6–9 appeared to contain only the smaller class of srPSTVd. Two size classes of srPSTVd were once again visible in the lower, fully expanded leaves 2–4 (data not shown; results similar to those shown in Fig. 1a). It would appear that once RNA silencing is triggered in PSTVd-infected plants, short srPSTVd containing ca 21 nt quickly accumulates to detectable levels in all above-ground portions of the plant, except the apical meristem.
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The time-course of symptom development and recovery for cucumber seedlings inoculated with HSVd at the cotyledon stage was very similar to that observed for PSTVd-infected tomato. Thirty d.p.i., pooled tissue samples from leaves 1–18 were assembled from ten plants and analysed for the presence of srHSVds. As shown in Fig. 2(b), the older portions of the cucumber plants (leaves 4–14) contained two different sizes of srHSVds; in the youngest leaves, the upper band was very faint. Overall, the distribution pattern of these two size classes of srHSVd was quite similar to that observed for srPSTVd in tomato.
Field-grown hops infected with either HLVd alone or mixtures of HLVd+HSVd or HLVd+AFCVd were also examined for the presence of the respective Vd-sRNAs. As shown in Fig. 2(c), small RNAs derived from all three viroids were easily detectable in mature hops. Comparison of the left and right panels reveals that infection with either HSVd or HLVd led to the accumulation of two size classes of Vd-sRNA. In contrast, plants infected with HLVd+AFCVd contained relatively little of the longer srAFCVd (middle panel). Although it was not possible to follow accumulation patterns under field conditions in detail, at least two out of three viroids infecting hops appeared to give rise to two classes of small RNA. For purposes of comparison, we also examined the size class of small RNAs associated with CMV, a tripartite RNA virus replicating in the cytoplasm. Data presented in Fig. 2(d) show that, under the same experimental conditions used for PSTVd-infected plants, fully expanded lower leaves contained only short small RNA 14 d.p.i. No longer small RNAs were detectable.
As described in the Supplementary Materials, available with the online version of this paper, cDNAs derived from electophoretically purified srPSTVds were amplified, hybrid-selected using biotinylated PSTVd negative-strand RNA as capture probe (Tschudi et al., 2003), and cloned for sequence analysis. Purified srPSTVds containing shorter and longer size classes of small RNAs were prepared from the first symptomatic leaf (i.e. leaf 3) of tomato plants harvested approximately one week after symptoms had begun to appear (i.e. 20 d.p.i.). A comparable preparation of srPSTVd from the still-unfolding leaf 8 was also analysed.
As shown in Fig. 3(a), the 89 srPSTVd sequences recovered from leaf 3 were derived from the left and right terminal domains plus the border between the lower portions of the variable and central domains. In contrast to the results recently reported by Itaya et al. (2007), many srPSTVds were derived from PSTVd negative-strand RNA. Such srPSTVds were particularly common among the molecules originating from the border between the variable and central domains. A similar clustering was observed for the 85 srPSTVds recovered from leaf 8 (results not shown).
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compare the length distributions for the srPSTVd populations recovered from leaves 3 and 8. Data for srPSTVds derived from both the positive- and negative-strand PSTVd RNAs are presented, and two conclusions are immediately apparent. First, the srPSTVds recovered from leaf 8 appear to be more homogeneous in length than those from leaf 3; second, comparatively few 24 nt srPSTVds were recovered from either sample. Taken together, these results suggest that the mechanism responsible for srPSTVds biogenesis may well be more complicated than previously believed. Following the establishment of PSTVd-induced RNA silencing in tomato plants, two size classes of srPSTVds were detectable. Shorter molecules containing ca 21 nt were the first to appear, and longer ones ca 24 nt in length appeared several days later. A similar pattern was observed in cucumber plants infected with HSVd and field-grown hops infected with HLVd or doubly infected with HLVd plus HSVd or AFCVd. Analysis of leaf tissue collected from CMV-infected tomato, in contrast, revealed quite a different picture. No long srCMV could be detected in any of the leaves, even in fully expanded symptomatic leaves, and thus, the pattern of small RNA accumulation in which synthesis of short 21–22 nt small RNAs is followed several days later by the synthesis of longer molecules containing ca 24 nt appears to be unique to viroids that replicate in the host cell nucleus.
This chronology suggests a possible process by which RNA silencing reduces the severity of PSTVd disease while leaving replication relatively unaffected; namely, (i) immediately after induction of silencing, srPSTVd spreads systemically in the plant, (ii) leaf primordia developing thereafter are exposed to the RNA silencing from the beginning of the differentiation process and (iii) a marked suppression of visible disease becomes visible in the upper portions of infected plants as these leaves expand over the next two weeks. Even though PSTVd titres are somewhat lower in leaves exhibiting this recovery phenomenon (Sano & Matsuura, 2004), PSTVd accumulation remains relatively high up to 30 d.p.i. Unlike potato virus X and several other RNA viruses in which the onset of RNA silencing leads to a dramatic drop in viral RNA accumulation, PSTVd replication appears to be relatively resistant to RNA silencing (Wang et al., 2004; Itaya et al., 2007).
Sequence analysis of srPSTVd populations isolated from tomato leaves of different ages indicated that more than half of such molecules were derived from non-genomic PSTVd RNA(s), either from double-stranded molecules such as replicative intermediates or single-stranded negative-strand RNA. Both positive- and negative-strand PSTVd RNAs are known to assume highly base-paired dsRNA-like conformations (e.g. Steger & Riesner, 2003), and it is likely that either could serve as substrate for srPSTVd synthesis, as has been reported for the genomic RNA of certain plant viruses (Molnár et al., 2005; Ho et al., 2006). Indeed, Itaya et al. (2007) have recently presented evidence that (i) srPSTVd are predominantly derived from the positive-strand genomic RNA and (ii) incubation of full-length, linear positive-strand PSTVd RNA with a partially purified preparation of DICER-LIKE enzymes (DCLs) from Arabidopsis releases small RNAs containing 
21 nt. Significantly, two of the hotspots for srPSTVd biogenesis revealed by our analyses (i.e. those in the right and left terminal domains) are the same as those reported by Itaya et al. (2007).
The most striking difference between our respective datasets involves a third srPSTVd hotspot located between the central and variable domains on the lower strand of PSTVd. Here, a large majority of srPSTVd was derived from the negative rather than the positive strand. More extensive sequencing studies will be required to completely resolve this apparent discrepancy in srPSTVd origin, but we note that interaction of nearby positions 227–236 and 319–328 results in the formation of so-called ‘secondary hairpin II’. Mutational analysis of PSTVd secondary hairpin II suggests that it plays a critical role in negative-strand function in vivo (Loss et al., 1991), and perhaps one or more DCLs also recognize this alternative structural feature. It is also possible that sampling differences may be responsible for some of the differences observed. For example, our srPSTVd samples were extracted from still-expanding leaves 20 d.p.i., while Itaya et al. (2007) used a combination of new and expanding leaves collected 25–30 d.p.i. (Asuka Itaya, personal communication). As noted above, srPSTVd accumulation patterns were seen to differ dramatically depending on the stage of leaf development, and evidence was obtained for a time-dependent increase in srPSTVd size.
According to currently accepted models for small RNA biogenesis (e.g. Tang et al., 2003; Hamilton et al., 2002; Papp et al., 2003; Vaucheret 2006), different DCLs are involved in the synthesis of different size classes of small RNAs. The fact that plants infected with pospiviroids contain both long and short small RNAs strongly suggests the involvement of two or more DCLs. It is possible that PSTVd replicative intermediates located in the nucleus are processed by the tomato counterparts of DCL-1, -3 or -4 into small 19–22 nt RNAs which are then exported into the cytoplasm (Denti et al., 2004) via pathway(s) involving HASTY and/or other nucleocytoplasmic transport receptors (Park et al., 2005). Later, the large amounts of PSTVd progeny which accumulate in the nucleus/nucleolus could be processed by DCL-3 (or another DCL) to produce longer srPSTVd. Xie et al. (2004) have shown that DCL-3 is localized in the nucleus, and several other reports have shown that knocking out DCL-3 abolishes the synthesis of 24 nt small RNA in Arabidopsis (reviewed by Herr, 2005; Qi & Hannon, 2005). According to this scenario, the failure of CMV to enter the nucleus would protect it against degradation by DCL-3, thereby blocking the synthesis of longer small RNAs.
A final level of complexity may be due to the terminal methylation that is commonly observed for plant small RNA species (Yu et al., 2005; Ebhardt et al., 2005; Yang et al., 2006). In our analyses, the cloning efficiency of longer srPSTVd containing 23–24 nt was quite low, in spite of the intense hybridization signals observed in Northern analysis. Recently, Pak & Fire (2007) and Sijen et al. (2007) have shown that the secondary short interfering RNAs produced by RNA-directed RNA polymerases in Caenorhabditis elegans contain 5'-di- or triphosphate termini, resulting in a dramatic decrease in cloning efficiency. Pak & Fire (2007) further noted that (i) the cloning efficiency of small antisense RNAs could be increased by using a 5'-ligation-independent cloning method and (ii) small antisense RNAs cloned with a 5'-ligation-independent method were 1 nt longer than those cloned with a 5'-ligation-dependent method. Since our studies relied on a 5'-ligation-dependent method, the lower cloning efficiency of longer srPSTVd could be explained by the cloning method used. The longer srPSTVds observed in this research could represent secondary small RNAs with similar di- or triphosphate termini.
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ACKNOWLEDGEMENTS |
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Received 9 June 2007;
accepted 10 August 2007.
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