Modification of small RNAs associated with suppression of RNA silencing by tobamovirus replicase protein

Abstract

Plant viruses act as triggers and targets of RNA silencing and have evolved proteins to suppress this plant defense response during infection. Although Tobacco mosaic tobamovirus (TMV) triggers the production of virus-specific small interfering RNAs (siRNAs), this does not lead to efficient silencing of TMV nor is a TMV-green fluorescent protein (GFP) hybrid able to induce silencing of a GFP-transgene in Nicotiana benthamiana, indicating that a TMV silencing suppressor is active and acts downstream of siRNA production. On the other hand, TMV-GFP is unable to spread into cells in which GFP silencing is established, suggesting that the viral silencing suppressor cannot revert silencing that is already established. Although previous evidence indicates that the tobamovirus silencing suppressing activity resides in the viral 126-kDa small replicase subunit, the mechanism of silencing suppression by this virus family is not known. Here, we connect the silencing suppressing activity of this protein with our previous finding that Oilseed rape mosaic tobamovirus infection leads to interference with HEN1-mediated methylation of siRNA and micro-RNA (miRNA). We demonstrate that TMV infection similarly leads to interference with HEN1-mediated methylation of small RNAs and that this interference and the formation of virus-induced disease symptoms are linked to the silencing suppressor activity of the 126-kDa protein. Moreover, we show that also Turnip crinkle virus interferes with the methylation of siRNA but, in contrast to tobamoviruses, not with the methylation of miRNA.

FIG. 1.

TMV does not suppress a preestablished silencing system in N. benthamiana. (A and B) Spread of TMV-GFP in GFP-transgenic line 16c. The virus spreads freely throughout the GFP-expressing leaf (A, 8 dpi; B, 20 dpi). (C) TMV-GFP does not infect fully GFP-silenced leaves. (D) Fully GFP-silenced leaf infected with TMV-luc. (E and F) The spread of TMV-GFP in a leaf undergoing the spread of GFP silencing (E, 4 dpi; F, 15 dpi). Infection is restricted to nonsilenced areas of the leaf, indicating that TMV-GFP can propagate in GFP-expressing cells but is a target for silencing in GFP-silenced cells. Magnified images of leaf areas indicated in panels E and F are shown. Because the virus was unable to spread into cells undergoing GFP silencing, infection sites developed aberrant shapes.

FIG. 2.

An amino acid exchange mutation in the 126k/183k replicase protein interferes with silencing suppressing activity. (A) Position of the C349Y exchange mutation in ToMV and TMV. (B) Infection sites (7 dpi) of TMV-126k^wt-GFP (TMV-GFP) appear in the form of green fluorescent disks. (C) Infection sites (7 dpi) of TMV-126k^m-GFP (TMV-GFP carrying the C349Y mutation) appear in the form of green fluorescent rings, thus indicating silencing of the virus in cells behind the infection front. (D) Transient agroinfiltration assay in GFP-transgenic line 16c to determine silencing suppressor activity of the TMV 126k protein. GFP-expressing construct (p35S:GFP) was agroinfiltrated together with test construct encoding the TMV 126k protein (126k^wt), or the 126k protein carrying the C349Y mutation (126k^m), or the PVY Hc-Pro or with empty vector. The results shown were obtained at 5 dpi. Consistent with the strong ability of potyviral Hc-Pro to suppress silencing, the coexpression of this protein with the GFP construct results in strong expression of GFP. Coexpression of the GFP construct with empty control vector results in silencing of GFP. Expression of the TMV 126k protein (126k^wt) allows some expression of GFP, indicating the ability of the protein to suppress the silencing of GFP expression. In contrast, expression of 126k^m results in only very little GFP expression, indicating that the mutation reduces the silencing suppressing activity of 126k protein.

FIG. 3.

Phenotypes of tobacco plants and leaves after infection. (A and B) TMV infection led to severely stunted plants, a dark green appearance, and green mosaic leaves with irregular shape. (E to F) Systemic leaves displayed all patterns of deformation, ranging from an altered length/width ratio (E) over lancet-shaped leaves with distorted vein patterns (F) to extremely reduced leaf blades (G). (C and D) In contrast, infection with TMV-126k^m caused only minor symptoms, i.e., the growth was slightly inhibited (C) and the leaves had an almost-normal appearance, except that they appeared lighter green compared to the mock-infected controls (D). (H and I) The mutated virus could spread systemically throughout the plant, as was indicated by the mosaic pattern of systemic leaves.

FIG. 4.

sRNA analysis before (−) and after (+) β elimination (BE). ORMV infection causes the production of nonmethylated sRNA (viral siRNAs and indicated miRNAs) in Arabidopsis (A.t.), N. benthamiana (N.b.), and N. tabacum (N.t). TMV gives rise to the production of nonmethylated sRNA in N. tabacum like ORMV. The fraction (%) of β-elimination-sensitive nonmethylated miRNA within the total specific miRNA population is given for the TMV-infected samples. Compared to the fraction of nonmethylated miRNA observed in plants infected with wild-type TMV (column a), the fraction of nonmethylated miRNAs is reduced by ca. 50% in plants infected with TMV-126k^m (column b). In uninfected control tobacco plants, the tested miRNAs are fully methylated.

FIG. 5.

The level of nonmethylated sRNAs in TMV-infected plants correlates with the time course of infection and 126k expression. Comparison of RNA (A, B, C, D, and E) and protein (F and G) extracts derived from N. tabacum plants inoculated with wild-type TMV (W) or TMV-126k^m (M) or mock infected (C) and then harvested at 8, 15, 30, or 40 dpi. Viral siRNA (CP siRNA), shown before (−) (A) and after (+) (B) β elimination (BE), accumulates until about 30 dpi and shows a lower level at 40 dpi, suggesting protection of the virus genome by encapsidation. The virus-induced accumulation of nonmethylated siRNA (B) and miRNA (miR160) (C) is restricted to early time points (until 30 dpi) and correlated with the level of 126k/183k protein (F), as detected by immunoblotting. The level of virus-induced nonmethylated sRNA is reduced in extract derived from plants infected with TMV-126k^m (M); the effects of the suppressor mutation are more pronounced on miR160 (C) than on viral siRNA (B) and more pronounced during earlier time points (8 and 15 dpi) compared to later time points (30 dpi) (C). miR160 is fully methylated in uninfected control plants (lanes C). The level of TMV RNA (D) is reduced at 30 dpi, a finding consistent with high levels of CP siRNA (A), and increased at 40 dpi (D), a finding consistent with reduced levels of siRNAs (A) and protection of the viral genome by encapsidation. (E) Mitochondrial RPL2 mRNA is shown as RNA loading control; (F) immunoblot with antibody against TMV replicase; (G) bands in Coomassie blue-stained protein gel shown as loading control; (H) quantified fraction (%) of nonmethylated CP siRNA and miR160 within the total respective sRNA population in extracts derived from plants infected with wild-type TMV (W) and mutant TMV (TMV-126k^m, M), respectively. Also, the fraction (%) of nonmethylated sRNA accumulation seen in tissues infected by the mutant virus (M) compared to nonmethylated sRNA accumulation seen in tissues infected by the wild-type virus (W) (set to 100%) is shown.

FIG. 6.

Accumulation of nonmethylated miRNA in agroinfiltrated N. tabacum leaves expressing 126k protein. (A) Compared to TMV-infected leaves (TMV), the agroinfiltrated, 126k-transfected leaves (126k) express only very low levels of detectable 126k protein. Nevertheless, RNA samples treated (+) or not treated (−) for β elimination (BE) reveal that, similar to the infected leaves (TMV), 126k-transfected leaves also accumulate periodate-sensitive, nonmethylated miRNAs (arrow), whereas mock-infected or mock-transfected leaves (C) only accumulate periodate-insensitive and, therefore, fully methylated miRNA (see panel B).

FIG. 7.

In Arabidopsis, both ORMV and TCV infection lead to the accumulation of nonmethylated viral siRNA, but only ORMV infection leads also to the accumulation of nonmethylated miRNA. A. Analysis of miR166 (a), miR172 (b), TCV siRNA (with homology to the CP gene (c), ORMV siRNA (with homology to the CP gene) (d), and ORMV genomic RNA (e) in A. thaliana Col-0 extracts obtained at 14, 21, or 30 dpi from plants infected with either TCV or ORMV (OR) or from uninfected control plants (C). The sRNA extracts have been treated (+) or not treated (−) for β elimination (BE). The miRNAs are fully methylated in uninfected control plants (C), as seen by the stability of the miRNA and the absence of miRNA derivatives of higher (due to polyuridinylation) or lower molecular weight in the BE samples (a and b). Such miRNA derivatives are present in samples from ORMV-infected plants but not in samples from TCV-infected samples (a and b), indicating the accumulation of nonmethylated miRNA in ORMV-infected plants but not in TCV-infected plants. Viral siRNA is sensitive to BE, irrespective of whether the siRNA originates from TCV (c) or ORMV (d). B. Duplicate experiment. sRNAs extracted at 16 dpi from A. thaliana Col-0 and Ler ecotypes infected with TCV and either treated (+) or not treated (−) for β elimination (BE) were hybridized with probes against miR165 (a) and TCV siRNA (b). Whereas miRNA is fully methylated (a), a considerable fraction of the viral siRNA is nonmethylated (b).