Defective interfering RNA hinders the activity of a tombusvirus-encoded posttranscriptional gene silencing suppressor

Abstract

Defective interfering (DI) RNAs are subviral replicons originating from the viral genome and are associated with many plant RNA viruses and nearly all animal RNA viruses. The presence of DI RNAs in tombusvirus-infected plants reduces the accumulation of helper virus RNA and results in the development of attenuated symptoms similar to those caused by tombusviruses defective in p19, the posttranscriptional gene silencing (PTGS) suppressor. In situ analysis of infected plants containing DI RNAs revealed that the extent of virus infection was spatially restricted as was found for p19-defective tombusvirus. Previously, p19 was shown to suppress PTGS by sequestering the small interfering RNAs (siRNAs), which act as the specificity determinant for PTGS. Our results demonstrate that DI RNAs dramatically elevate the level of virus-specific siRNAs in viral infections, resulting in the saturation of p19 and the accumulation of unbound siRNAs. Moreover, we showed that, at low temperature, where PTGS is inhibited, DI RNAs are not able to efficiently interfere with virus accumulation and protect the plants. These data show that the activation of PTGS plays a pivotal role in DI RNA-mediated interference. Our data also support a role for 21-nucleotide siRNAs in PTGS signaling.

FIG. 1.

Accumulation of virus and DI RNAs in systemically infected leaves of N. benthamiana at 7 dpi. (A) Schematic representation of the organization of the TBSV-P genome and DI RNAs. RdRp, RNA-dependent RNA polymerase; MP, movement protein. (B) Northern blot analysis of TBSV-P, T19stop, and TBSV-P+DI accumulation in systemically infected leaves. Relative gel loadings are shown by ethidium bromide staining of rRNAs (bottom). G, genomic RNA; sg, sg RNA; DI, DI RNA. (C to E) In situ hybridization of leaf cross sections at 7 dpi. (C) TBSV-P-infected tissue. (Inset) Control mock-inoculated tissue. (D) T19stop-infected tissue. (E) TBSV-P+DI-infected tissue. The bar in panel C applies also to panels D and E. Black triangles, sites of virus accumulation.

FIG. 2.

Accumulation of viral RNA and p19 in TBSV-P- and TBSV-P+DI-transfected protoplasts and infected tissues. (A) Northern analyses of helper virus accumulation in transfected protoplasts at different temperatures at 24 and 48 hpt. (B) Western blot analyses of p19 accumulation in transfected protoplasts in two independent samples at 24°C. (C) In situ hybridization of leaf cross sections detecting the accumulation of plus-sense viral RNA at 7 dpi. An antisense (as) CP RNA probe detects the genomic RNAs but not DI RNAs. The color reactions of nearly consecutive sections of TBSV-P-, TBSV-P+DI-, and mock-inoculated tissues were halted at different time points before reaching the saturation level. The bar applies to all images in panels C and D. (D) Immunohistochemistry applying an anti-p19 antibody to nearly consecutive sections of TBSV-P-, TBSV-P+DI-, and mock-inoculated tissues. The color reactions were stopped at the times indicated.

FIG. 3.

Accumulation of virus-specific siRNAs and p19 in the systemically infected leaves at 7 dpi. (A, top) Northern blot analysis of the accumulation of genomic and DI RNAs in TBSV-P- and TBSV-P+DI-infected plants. (Middle) Accumulation of p19 in the corresponding samples. (Bottom) siRNA accumulation in the corresponding samples, as shown with a genomic-RNA-specific probe. The calculated siRNA/p19 ratios are indicated at the bottom. (B) Fractionation of crude extracts prepared from TBSV-P-, T19stop-, and TBSV-P+DI-infected plants with a Superdex-200 gel filtration column. Collected fractions were tested for the presence of virus-specific siRNAs and p19 by Northern and Western blot analyses. A γ-ATP-labeled 21-nt synthetic RNA oligonucleotide was used as a size marker. (C) Extracts prepared from leaves of TBSV-P-, T19stop-, TBSV-P+DI-, and mock-inoculated plants were immunoprecipitated with an anti-p19 antibody. Immunoprecipitates were analyzed for the accumulation of p19 and virus-specific siRNAs by Western and Northern blot analyses.

FIG. 4.

Effect of temperature on DI RNA-containing-TBSV-P infection. (A) Northern blot analysis of TBSV-P- and TBSV-P+DI-infected plants. Samples were taken at 7 and 14 dpi at 15, 21, and 24°C as indicated. Mock, mock-inoculated tissue. (B) Symptoms induced by TBSV-P and TBSV-P+DI infections at 15, 21, and 24°C at 14 dpi. (C) TBSV-P genomic RNA accumulation in systemically infected leaves at 7 and 14 dpi. Sections were hybridized with a CP ORF-specific RNA probe. Samples and the applied temperatures are indicated.

FIG. 5.

Proposed model for the PTGS-based mechanism of DI RNA-mediated interference. (A) In wild-type virus infections, the viral genomic RNA accumulates to high levels in infected mesophyll cells and the PTGS machinery of the host produces virus-specific siRNAs. However, at the same time, the virus translates p19, which physically binds the generated siRNAs, inhibiting their spread out of the infected cell. Consequently the cells ahead the infection front remained unprotected against the spreading virus. (B) Cells infected with p19-defective virus accommodate the same level of virus genomic RNA as cells infected with wild-type virus because RNA silencing has no capacity to cope with the invasive accumulation of virus-derived products. However, the absence of p19 results in the accumulation of free virus-specific siRNAs, which are able to traffic through plasmodesmata to cells beyond the infection front. These virus-specific mobile siRNAs are incorporated into the RISC of cells ahead of the infection front. Thus the already-activated RISCs destroy the entering viral RNA before the establishment of virus replication. (C) In the presence of DI RNAs, the infected cells accumulate significant amounts of genomic RNA and p19. However, because DI RNAs are poor targets of PTGS, they accumulate to extremely high levels and provide a source for the generation of extra amounts of virus-specific siRNAs, saturating the p19-binding capacity. The remaining free siRNAs are able to induce a similar process, which has been described for p19-defective-virus infection. Other previously described DI RNA-associated factors, such as suppressed rate of accumulation of genomic RNA and reduced p19 accumulation in early stages of virus infection, may further enhance the efficiency of PTGS-based defense by increasing the amount of free siRNA. The synergistic mode of action of different factors results in restricted spread of the virus and development of attenuated symptoms.