Characterization of the siRNAs associated with peach latent mosaic viroid infection

St-Pierre, Patrick; Hassen, I. Fekih; Thompson, Dan; Perreault, Jonathan

doi:10.1016/j.virol.2008.11.008

Cited by 40 publications

(39 citation statements)

References 19 publications

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“…Infection by viroids from both families occurs with the accumulation of 21-to 24-nt viroid-derived small RNAs (vd-sRNAs) with the characteristic properties of the siRNAs associated with RNA silencing, as revealed by RNA gel blot hybridization (5,36,43,46,47,59,74), low-scale sequencing (35,41,44,77), and deep sequencing (17,52). Moreover, mechanically codelivered or agroinoculated dsRNAs interfere with viroid infection in a sequence-specific manner (as also do, at least in one case, in vitro-generated homologous vd-sRNAs codelivered mechanically) (5).…”

mentioning

confidence: 99%

RNA-Dependent RNA Polymerase 6 Delays Accumulation and Precludes Meristem Invasion of a Viroid That Replicates in the Nucleus

Serio¹,

Alba

Navarro³

et al. 2010

J Virol

143

101

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The detection of viroid-derived small RNAs (vd-sRNAs) similar to the small interfering RNAs (siRNAs, 21 to 24 nucleotides [nt]) in plants infected by nuclear-replicating members of the family Pospiviroidae (type species, Potato spindle tuber viroid [PSTVd]) indicates that they are inducers and targets of the RNA-silencing machinery of their hosts. RNA-dependent RNA polymerase 6 (RDR6) catalyzes an amplification circuit producing the double-stranded precursors of secondary siRNAs. Recently, the role of RDR6 in restricting systemic spread of certain RNA viruses and precluding their invasion of the apical growing tip has been documented using RDR6-silenced Nicotiana benthamiana (NbRDR6i) plants. Here we show that RDR6 is also engaged in regulating PSTVd levels: accumulation of PSTVd genomic RNA was increased in NbRDR6i plants with respect to the wild-type controls (Nbwt) early in infection, whereas this difference decreased or disappeared in later infection stages. Moreover, in situ hybridization revealed that RDR6 is involved in restricting PSTVd access in floral and vegetative meristems, thus providing firm genetic evidence for an antiviroid RNA silencing mechanism. RNA gel blot hybridization and deep sequencing showed in wt and RDR6i backgrounds that PSTVd sRNAs (i) accumulate to levels paralleling their genomic RNA, (ii) display similar patterns with prevailing 22-or 21-nt plus-strand species, and (iii) adopt strand-specific hot spot profiles along the genomic RNA. Therefore, the surveillance mechanism restraining entry of some RNA viruses into meristems likely also controls PSTVd access in N. benthamiana. Unexpectedly, deep sequencing also disclosed in NbRDR6i plants a profile of RDR6-derived siRNA dominated by 21-nt plus-strand species mapping within a narrow window of the hairpin RNA stem expressed transgenically for silencing RDR6, indicating that minus-strand siRNAs silencing the NbRDR6 mRNA represent a minor fraction of the total siRNA population.Viroids are a unique class of replicons that in their minimal genome-composed exclusively of a circular, non-protein-coding single-stranded RNA (ssRNA) of only 246 to 401 nucleotides (nt), which folds into a compact secondary structurestore enough genetic information to initiate the infection of certain plants, to usurp their gene expression machinery for producing progenies that spread systemically, and to induce frequently specific diseases (14,15,16,22,23,81). The available evidence indicates that viroids replicate in the nucleus (family Pospiviroidae) or in the chloroplast (family Avsunviroidae) through a rolling-circle mechanism involving RNA intermediates, some of which form double-stranded-RNA (dsRNA) complexes (3,4,9,21,53). In the type species of the family Pospiviroidae, Potato spindle tuber viroid (PSTVd) (55), the infecting monomeric circular RNA, the mc plus strand (Fig. 1), is reiteratively transcribed into oligomeric minusstrand RNA intermediates that are then transcribed into oligomeric plus-strand RNAs and finally cleaved and ligated into the mc ...

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mentioning

confidence: 99%

RNA-Dependent RNA Polymerase 6 Delays Accumulation and Precludes Meristem Invasion of a Viroid That Replicates in the Nucleus

Serio¹,

Alba

Navarro³

et al. 2010

J Virol

143

101

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“…Each preparation was linked to a bar-coded adapter in order to sequence the two libraries in the same channel, thus avoiding any undesired bias and obtaining two independent datasets that could be further compared. Moreover, PLMVd-sRNA analysis was simplified because the progenies resulting from the two specific PLMVd variants, which come from greenhouse plants that have been infected for a short time, are considerably less complex populations than those from field trees [26] that may have been infected (and even re-infected) for a long time. Additionally, we had previous information about the sequence of eight variants from the GDS6 progeny [32] and of 16 variants from the PC-C40 progeny (Navarro et al, unpublished data), which was used in the subsequent analysis.…”

Section: Resultsmentioning

confidence: 99%

“…Adapters were synthesized assuming that the vd-sRNAs have the characteristic 5′ phosphomonoester and 3′ hydroxyl termini resulting from DCL activity [17], [18]. Although earlier data indicate that this maybe the case [25], [26], further studies are needed to exclude the possibility that a fraction of the vd-sRNAs may have other 5′ termini as recently reported for secondary siRNAs in Caenorhabditis elegans [38], [39].…”

Section: Resultsmentioning

confidence: 99%

“…Moreover, low-scale sequencing of the vd-sRNAs from two members of the family Pospiviroidae [23]–[25] and very recently of one of the Avsunviroidae [26] has confirmed that they are predominantly of 21 and 22 nt and (+) polarity, although there are some discrepancies even between results derived from the same viroid-host combination —with one report describing an abundant cluster of (−) sRNAs [24]— that may be in part the consequence of the limited dataset generated by low-scale sequencing.…”

Section: Introductionmentioning

confidence: 99%

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Deep Sequencing of the Small RNAs Derived from Two Symptomatic Variants of a Chloroplastic Viroid: Implications for Their Genesis and for Pathogenesis

et al. 2009

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Northern-blot hybridization and low-scale sequencing have revealed that plants infected by viroids, non-protein-coding RNA replicons, accumulate 21–24 nt viroid-derived small RNAs (vd-sRNAs) similar to the small interfering RNAs, the hallmarks of RNA silencing. These results strongly support that viroids are elicitors and targets of the RNA silencing machinery of their hosts. Low-scale sequencing, however, retrieves partial datasets and may lead to biased interpretations. To overcome this restraint we have examined by deep sequencing (Solexa-Illumina) and computational approaches the vd-sRNAs accumulating in GF-305 peach seedlings infected by two molecular variants of Peach latent mosaic viroid (PLMVd) inciting peach calico (albinism) and peach mosaic. Our results show in both samples multiple PLMVd-sRNAs, with prevalent 21-nt (+) and (−) RNAs presenting a biased distribution of their 5′ nucleotide, and adopting a hotspot profile along the genomic (+) and (−) RNAs. Dicer-like 4 and 2 (DCL4 and DCL2, respectively), which act hierarchically in antiviral defense, likely also mediate the genesis of the 21- and 22-nt PLMVd-sRNAs. More specifically, because PLMVd replicates in plastids wherein RNA silencing has not been reported, DCL4 and DCL2 should dice the PLMVd genomic RNAs during their cytoplasmic movement or the PLMVd-dsRNAs generated by a cytoplasmic RNA-dependent RNA polymerase (RDR), like RDR6, acting in concert with DCL4 processing. Furthermore, given that vd-sRNAs derived from the 12–14-nt insertion containing the pathogenicity determinant of peach calico are underrepresented, it is unlikely that symptoms may result from the accidental targeting of host mRNAs by vd-sRNAs from this determinant guiding the RNA silencing machinery.

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“…De manera similar se han identificado y caracterizado vd-sRNAs en plantas infectadas por los viroides cloroplásticos PLMVd, CChMVd y ASBVd (Martínez de Alba et al, 2002;Markarian et al, 2004;Di Serio et al, 2009;St-Pierre et al, 2009;Bolduc et al, 2010). Puesto que no se han descrito procesos de PTGS que ocurran en el cloroplasto, se asume que dichos vd-sRNAs son generados en el citoplasma durante el movimiento célula a célula del RNA viroidal, una posibilidad que puede extenderse a los viroides nucleares.…”

Section: El Silenciamiento Génico En El Contexto De La Infección Virounclassified

Caracterización de actividades RNA ligasa implicadas en la replicación de los viroides nucleares y cloroplásticos

Zafra¹,

Ángeles²

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Characterization of the siRNAs associated with peach latent mosaic viroid infection

Cited by 40 publications

References 19 publications

RNA-Dependent RNA Polymerase 6 Delays Accumulation and Precludes Meristem Invasion of a Viroid That Replicates in the Nucleus

RNA-Dependent RNA Polymerase 6 Delays Accumulation and Precludes Meristem Invasion of a Viroid That Replicates in the Nucleus

Deep Sequencing of the Small RNAs Derived from Two Symptomatic Variants of a Chloroplastic Viroid: Implications for Their Genesis and for Pathogenesis

Caracterización de actividades RNA ligasa implicadas en la replicación de los viroides nucleares y cloroplásticos

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