Deep sequencing of viroid-derived small RNAs from grapevine provides new insights on the role of RNA silencing in plant-viroid interaction

Abstract

Background: Viroids are circular, highly structured, non-protein-coding RNAs that, usurping cellular enzymes and escaping host defense mechanisms, are able to replicate and move through infected plants. Similarly to viruses, viroid infections are associated with the accumulation of viroid-derived 21-24 nt small RNAs (vd-sRNAs) with the typical features of the small interfering RNAs characteristic of RNA silencing, a sequence-specific mechanism involved in defense against invading nucleic acids and in regulation of gene expression in most eukaryotic organisms.

Methodology/principal findings: To gain further insights on the genesis and possible role of vd-sRNAs in plant-viroid interaction, sRNAs isolated from Vitis vinifera infected by Hop stunt viroid (HSVd) and Grapevine yellow speckle viroid 1 (GYSVd1) were sequenced by the high-throughput platform Solexa-Illumina, and the vd-sRNAs were analyzed. The large majority of HSVd- and GYSVd1-sRNAs derived from a few specific regions (hotspots) of the genomic (+) and (-) viroid RNAs, with a prevalence of those from the (-) strands of both viroids. When grouped according to their sizes, vd-sRNAs always assumed a distribution with prominent 21-, 22- and 24-nt peaks, which, interestingly, mapped at the same hotspots.

Conclusions/significance: These findings show that different Dicer-like enzymes (DCLs) target viroid RNAs, preferentially accessing to the same viroid domains. Interestingly, our results also suggest that viroid RNAs may interact with host enzymes involved in the RNA-directed DNA methylation pathway, indicating more complex scenarios than previously thought for both vd-sRNAs genesis and possible interference with host gene expression.

Figure 1. HSVd- and GYSVd1-sRNAs from grapevine tissues are prevalently of (−) polarity.

Histograms comparing the total reads and the ratio of (+) and (−) HSVd-sRNAs (A and C) and GYSVd1-sRNAs (B and D) obtained by deep sequencing from berry, tendril, flower and leaf grapevine tissues.

Figure 2. Size distribution of HSVd- and GYSVd1-sRNAs reveals prominent peaks of 21-, 22- and 24-nt species.

Histograms comparing the size distribution of 20–24-nt HSVd-sRNAs (A) and GYSVd1-sRNAs (B) in the different grapevine tissues.

Figure 3. Relative abundance of vd-sRNAs with different size and 5′ termini.

Histograms comparing the size distribution (20–24-nt) and nucleotide at the 5′ termini of total (A and D), (+) (B and E) and (−) (C and F) HSVd-sRNAs reads (left panels) and GYSVd1-sRNAs reads (right panels) from tendril.

Figure 4. Most HSVd- and GYSVd1-sRNAs derive from restricted regions of the genomic (+) and (−) RNAs.

Location of the 5′ termini and frequency of the HSVd-sRNAs (A) and GYSVd1-sRNAs (B) from tendril in their corresponding (+) and (−) genomic RNAs. Positives values correspond to vd-sRNAs of (+) polarity and negative values correspond to the vd-sRNAs of (−) polarity. Note that the scale is different in the panels and that the same numbers are used in the (+) polarity (5′→3′ orientation is from left to right) and in the (−) polarity (5′→3′ orientation is from right to left). For the location of the 5′ termini of vd-sRNAs we have considered the HSVd and GYSVd1 sequence variant with the accession numbers X06873 and GQ995473, respectively. The viroid sequences covered by vd-sRNAs bellowing to hotspots (HS1, HS2 and HS3 for HSVd and GY1, GY2 and GY3 for GYSVd1) are denoted by arrows whose sense indicates 5′→3′ orientation.

Figure 5. HSVd-sRNAs and GYSVd1-sRNAs do not cover the same viroid structural domains.

Sequence and computer-predicted secondary structure for the (+) strand of the HSVd (sequence variant X06873) (A) and the GYSVd1 (sequence variant GQ995473) (B), corresponding to the master and the consensus variants in the grapevine sequenced viroid populations, respectively. The viroid sequences covered by vd-sRNAs corresponding to hotspots (HS1, HS2 and HS3 for HSVd, and GY1, GY2 and GY3 for GYSVd1) are denoted by arrows whose sense indicates 5′→3′ orientation. The position of five structural domains proposed for PSTVd and closely-related viroids are indicated (P: pathogenic; V: variable; C: central; T1: terminal left; T2: terminal right), although no data on the functional properties of these regions in HSVd and GYSVd1 are available. The secondary structures were obtained by the program Mfold .

Figure 6. Specific size-classes vd-sRNAs may largely prevail at certain genomic positions.

Histograms comparing location of the 5′ termini, frequency and size distribution of (−) vd-sRNAs corresponding to the HSVd hotspot 3 (HS3) and recovered from the different grapevine tissues. Numbers are referred to HSVd sequence variant with the accession number X06873. 5′→3′ orientation is from right to left.