Specific argonautes selectively bind small RNAs derived from potato spindle tuber viroid and attenuate viroid accumulation in vivo

Abstract

The identification of viroid-derived small RNAs (vd-sRNAs) of 21 to 24 nucleotides (nt) in plants infected by viroids (infectious non-protein-coding RNAs of just 250 to 400 nt) supports their targeting by Dicer-like enzymes, the first host RNA-silencing barrier. However, whether viroids, like RNA viruses, are also targeted by the RNA-induced silencing complex (RISC) remains controversial. At the RISC core is one Argonaute (AGO) protein that, guided by endogenous or viral sRNAs, targets complementary RNAs. To examine whether AGO proteins also load vd-sRNAs, leaves of Nicotiana benthamiana infected by potato spindle tuber viroid (PSTVd) were agroinfiltrated with plasmids expressing epitope-tagged versions of AGO1, AGO2, AGO3, AGO4, AGO5, AGO6, AGO7, AGO9, and AGO10 from Arabidopsis thaliana. Immunoprecipitation analyses of the agroinfiltrated halos revealed that all AGOs except AGO6, AGO7, and AGO10 associated with vd-sRNAs: AGO1, AGO2, and AGO3 preferentially with those of 21 and 22 nt, while AGO4, AGO5, and AGO9 additionally bound those of 24 nt. Deep-sequencing analyses showed that sorting of vd-sRNAs into AGO1, AGO2, AGO4, and AGO5 depended essentially on their 5'-terminal nucleotides, with the profiles of the corresponding AGO-loaded vd-sRNAs adopting specific hot spot distributions along the viroid genome. Furthermore, agroexpression of AGO1, AGO2, AGO4, and AGO5 on PSTVd-infected tissue attenuated the level of the genomic RNAs, suggesting that they, or their precursors, are RISC targeted. In contrast to RNA viruses, PSTVd infection of N. benthamiana did not affect miR168-mediated regulation of the endogenous AGO1, which loaded vd-sRNAs with specificity similar to that of its A. thaliana counterpart. Importance: To contain invaders, particularly RNA viruses, plants have evolved an RNA-silencing mechanism relying on the generation by Dicer-like (DCL) enzymes of virus-derived small RNAs of 21 to 24 nucleotides (nt) that load and guide Argonaute (AGO) proteins to target and repress viral RNA. Viroids, despite their minimal genomes (non-protein-coding RNAs of only 250 to 400 nt), infect and incite disease in plants. The accumulation in these plants of 21- to 24-nt viroid-derived small RNAs (vd-sRNAs) supports the notion that DCLs also target viroids but does not clarify whether vd-sRNAs activate one or more AGOs. Here, we show that in leaves of Nicotiana benthamiana infected by potato spindle tuber viroid, the endogenous AGO1 and distinct AGOs from Arabidopsis thaliana that were overexpressed were associated with vd-sRNAs displaying the same properties (5'-terminal nucleotide and size) previously established for endogenous and viral small RNAs. Overexpression of AGO1, AGO2, AGO4, and AGO5 attenuated viroid accumulation, supporting their role in antiviroid defense.

FIG 1

(A and B) PSTVd infection of N. benthamiana has no significant effect on AGO1 or miR168 accumulation in the upper, noninoculated leaves (A) or in the agroinfiltrated leaves (B). Western blot analyses were performed with a rabbit polyclonal antibody against the N-terminal region of AGO1 from N. benthamiana and a goat anti-rabbit secondary antibody conjugated to horseradish peroxidase. Total proteins were separated by PAGE in 4 to 12% gels, and equal loading was assessed by the intensity of the large subunit of RubisCO after staining with Ponceau S. Northern blot hybridizations were carried out with a 5′-radiolabeled oligodeoxyribonucleotide complementary to miR168. Total RNAs were separated by denaturing PAGE in 17% gels, and equal loading was assessed by the intensity of tRNA after staining with ethidium bromide. (A) Samples were collected at 15 (lanes 1 and 5), 20 (lanes 2 and 6), 25 (lanes 3 and 7), and 30 (lanes 4 and 8) days p.i. (dpi). (B) Samples were collected at 4 (lanes 1 and 4), 6 (lanes 2 and 5), and 8 (lanes 3 to 6) days p.i. (C) Endogenous AGO1 loads vd-sRNAs during PSTVd infection of N. benthamiana. Aliquots of total sRNA (INPUT) and of the sRNA fraction immunoprecipitated with a rabbit polyclonal antibody against the N-terminal region of AGO1 from N. benthamiana (α-AGO1) (IP) were separated by denaturing PAGE in 17% gels and revealed by Northern blot hybridization with a radiolabeled riboprobe for detecting PSTVd plus strands. Lanes 1 and 4, mock-inoculated control; lanes 2, 3, 5, and 6, PSTVd-infected upper, noninoculated leaves collected at 25 days postinoculation. IPs were obtained with the antibody against AGO1 (lanes 4 and 6) or with a preimmune rabbit immunoglobulin fraction (IgG) (lane 5). Mock inoculations were performed with cultures of A. tumefaciens with a binary plasmid expressing GUS instead of the head-to-tail dimeric plus transcript of PSTVd. Accumulation of the PSTVd MC and ML forms was also examined in the RNA inputs after denaturing PAGE in 5% gels (upper gel). Equal loading was assessed by the intensity of the bands generated by the 5S RNA and tRNAs after staining with ethidium bromide.

FIG 2

(A) AGO1 and AGO2, but not AGO7 or AGO10, specifically bind certain vd-sRNAs. Shown are Northern blot hybridizations with a full-length radiolabeled riboprobe for detecting PSTVd plus total RNA strands (INPUT or IN) from mock- and PSTVd-inoculated N. benthamiana agroinfiltrated with cultures of A. tumefaciens with binary plasmids for expressing HA-tagged AGO1 (lanes 1 and 2), AGO2 (lane 3), AGO7 (lanes 7 and 9), and AGO10 (lane 11). A size marker was included in lane M. RNA immunoprecipitates (IP) generated with an anti-HA monoclonal antibody from the halos agroexpressing HA-tagged AGO1 (lanes 4 and 5), AGO 2 (lane 6), AGO7 (lanes 8 and 10), and AGO10 (lane 12) were similarly analyzed. (B) Other agroinfiltrated AGOs, apart from AGO1 and AGO2, also bind vd-sRNAs with different affinities. Shown are Northern blot hybridizations with a full-length radiolabeled riboprobe for detecting PSTVd plus strands of total RNAs (INPUT) from mock- and PSTVd-inoculated N. benthamiana agroinfiltrated with cultures of A. tumefaciens with binary plasmids for expressing HA-tagged AGO2 (lanes 1, 2, 9, and 10), AGO4 (lane 3), AGO5 (lane 4), AGO3 (lane 11), AGO6 (lane 12), and AGO9 (lane 13). RNA immunoprecipitates (IP) generated with an anti-HA monoclonal antibody from the halos agroexpressing HA-tagged AGO2 (lanes 5, 6, 14, and 15), AGO4 (lane 7), AGO5 (lane 8), AGO3 (lane 16, overexposed to make the band visible), AGO6 (lane 17), and AGO9 (lane 18) were similarly analyzed. RNAs were separated by denaturing PAGE in 17% gels, and equal loading was assessed by the intensity of tRNA after staining with ethidium bromide. Western blot analyses of total proteins from halos were carried out with the anti-HA monoclonal antibody following protein separation by PAGE in 4 to 12% gels; equal loading was assessed by the intensity of the large subunit of RubisCO after staining with Ponceau S. Mock inoculations were performed with cultures of A. tumefaciens with a binary plasmid expressing GUS instead of the head-to-tail dimeric transcript of PSTVd. In all cases, samples were processed 2 days after agroinfiltration of plants that were PSTVd infected, or mock inoculated, 19 days before.

FIG 3

Size distribution of PSTVd and plant sRNAs in total RNAs (INPUT) and immunoprecipitates (IP) from halos of PSTVd-infected N. benthamiana agroinfiltrated with cultures of A. tumefaciens with binary plasmids for expressing HA-tagged versions of AGO1, AGO2, AGO4, and AGO5 from A. thaliana. The histograms compare the distributions of 18- to 26-nt total sRNA reads. The IP fractions were generated with an anti-HA monoclonal antibody. Note that the scales are not identical in the different histograms and that the fraction of PSTVd-sRNAs could be higher considering that the viroid may not invade all cells.

FIG 4

Sorting of PSTVd-sRNAs into AGO1, AGO2, AGO4, and AGO5 mainly depends on their 5′-terminal nucleotides. The histograms display, in total RNA (INPUT) and in RNA immunoprecipates (IP), the fraction (%) of total reads corresponding to the 21-, 22-, and 24-nt PSTV-sRNAs with distinct 5′ termini.

FIG 5

AGO-loaded vd-sRNAs adopt hot spot distributions along the viroid genome that are specific for each of the four HA-tagged AGOs from A. thaliana agroexpressed in PSTVd-infected N. benthamiana. Shown are the locations and frequencies in the genomic PSTVd RNA of the 5′ termini of the plus-strand (positive values) and minus-strand (negative values) vd-sRNA reads per million (rpm) from total RNAs (Input) and from immunoprecìpitates (IP) generated with an anti-HA monoclonal antibody. Note that the same numbers are used in the plus polarity (the 5′-to-3′ orientation is from left to right) and in the minus polarity (the 5′-to-3′ orientation is from right to left).

FIG 6

Agroexpression of AGO1, AGO2, AGO4, and AGO5, but not of AGO7 or of GUS, attenuates viroid accumulation. (A and B) Northern blot hybridizations with a full-length radiolabeled riboprobe for detecting PSTVd plus strands of total RNAs from halos of mock- and PSTVd-inoculated N. benthamiana agroinfiltrated with cultures of A. tumefaciens with binary plasmids for expressing HA-tagged AGO1 (A, lanes 1, 4, and 5), AGO2 (A, lanes 6 and 7), AGO4 (B, lanes 1, 4, 5, and 6), AGO5 (B, lanes 7, 8, and 9), AGO7 (A, lanes 8 and 9, and B, lanes 10 and 11), and GUS (A and B, lanes 2 and 3). Mock inoculations were performed as indicated in the legend to Fig. 3. Total RNAs, extracted 2 days after agroinfiltration, were separated by denaturing PAGE in 5% gels, and equal loading was assessed by the intensity of tRNA after staining with ethidium bromide. Western blot analyses of total proteins from halos were carried out with the anti-HA monoclonal antibody following protein separation by PAGE in 4 to 12% gels; equal loading was assessed by the intensity of the large subunit of RubisCO after staining with Ponceau S. In all cases, samples were processed 2 days after agroinfiltration of plants that were PSTVd infected, or mock inoculated, 8 days before.

FIG 7

Analysis of vd-sRNAs in the IP versus the input generated by a polyclonal antibody against AGO1 from N. benthamiana reveals a clear enrichment in the IP of plus (A) and minus (B) vd-sRNAs of 21 and 22 nt (but not 24 nt) with a 5′-terminal U. IP enrichment or depletion was determined for each unique 21-, 22-, or 24-nt vd-sRNA as log₂ [(IP reads + 1)/(input reads + 1)] and plotted for each size class as the fraction (%) of unique vd-sRNA sequences enriched >2-fold (log₂ >1) or depleted >2-fold (log₂ < 1) in the IP compared.

FIG 8

AGO1-loaded vd-sRNAs adopt a hot spot distribution along the viroid genome in PSTVd-infected N. benthamiana. Shown are the locations and frequencies in the genomic PSTVd RNA of the 5′ termini of the plus-strand (positive values) and minus-strand (negative values) vd-sRNA reads per million (rpm) from total RNAs (INPUT) and from the immunoprecipitate (IP) generated with an anti-N. benthamiana AGO1 (Nb AGO1) polyclonal antibody. See the legend to Fig. 5 for details.