Double-stranded RNA-mediated interference with plant virus infection

Abstract

Double-stranded RNA (dsRNA) has been shown to play a key role as an inducer of different interference phenomena occurring in both the plant and animal kingdoms. Here, we show that dsRNA derived from viral sequences can interfere with virus infection in a sequence-specific manner by directly delivering dsRNA to leaf cells either by mechanical inoculation or via an Agrobacterium-mediated transient-expression assay. We have successfully interfered with the infection of plants by three viruses belonging to the tobamovirus, potyvirus, and alfamovirus groups, demonstrating the reliability of the approach. We suggest that the effect mediated by dsRNA in plant virus infection resembles the analogous phenomenon of RNA interference observed in animals. The interference observed is sequence specific, is dose dependent, and is triggered by dsRNA but not single-stranded RNA. Our results support the view that a dsRNA intermediate in virus replication acts as efficient initiator of posttranscriptional gene silencing (PTGS) in natural virus infections, triggering the initiation step of PTGS that targets viral RNA for degradation.

FIG. 1

Specific interference with PMMoV infection by dsRNA in a hypersensitive host. Response of N. tabacum cv. Xanthi nc to PMMoV (5 μg/ml) alone (left halves of leaves) or to a combination of PMMoV plus either 54-kDa-protein dsRNA (A), 54-kDa-protein antisense (As) RNA (B), TEV-HC dsRNA (C), or in vitro transcription buffer (D) (right halves of the leaves). Leaves were photographed at 5 dpi. Similar numbers of local lesions (arrowheads) were observed in both halves of the leaves in panels B, C, and D. No visible local response was observed in the half-leaf inoculated with PMMoV plus 54-kDa-protein dsRNA.

FIG. 2

dsRNA-mediated interference with PMMoV infection in a systemic host. (A) Specificity of interference with PMMoV infection by dsRNA. Northern blot analysis of total RNA extracted from inoculated (lanes 1 to 6) or uppermost systemic (lanes 7 to 13) leaves of N. benthamiana. Plants were mock inoculated or were inoculated with PMMoV (5 μg/ml) alone (−), with 54-kDa-protein dsRNA alone, or with PMMoV plus either 54-kDa-protein dsRNA, sense (S) 54-kDa-protein RNA, antisense (As) 54-kDa-protein RNA, or 54-kDa-protein cDNA, as indicated. Leaf tissues were harvested at 7 dpi, except for the samples in lanes 12 and 13, which were harvested at 21 dpi. The samples in lanes 2, 8, and 12 were taken from the same plant, which did not display symptoms of infection until its life cycle was completed. The sample in lane 13 was taken from another individual showing disease symptoms at 21 dpi. The 54-kDa-protein dsRNA used in the inoculum was run in lane 15 for comparison. (B) Time course analysis of dsRNA stability on plant leaves. The 54-kDa-protein dsRNA (10 μl, 0.62 μM) was inoculated on fully expanded leaves of N. benthamiana (three- to four-leaf stage). After the inoculated leaves had been washed with Triton X-100 (0.05%) for 30 min, RNA was extracted at the indicated times. Mock, RNA extracted from a mock-inoculated plant. (C) Interference with PMMoV infection by homologous dsRNAs. RNA was extracted from upper leaf tissue of plants inoculated with PMMoV (5 μg/ml) alone (−) or with PMMoV plus the indicated RNAs. (D) Interference seems to require a minimum length of dsRNA. RNA was extracted from inoculated (lanes 1 and 4) or upper (lanes 2, 3, 5, and 6) leaf tissue of plants infected with PMMoV (5 μg/ml) alone or with PMMoV plus 1/3 54-kDa-protein dsRNA at 7 (lanes 1, 2, 4 and 5) or 12 dpi (lanes 3 and 6). The 1/3 54-kDa-protein dsRNA used in the inoculum was run in lane 7 for comparison. Similar amounts (1 μg) of RNA samples were fractionated by 1% agarose gel electrophoresis in all panels, and a DIG-labeled 54-kDa-protein RNA was used as a probe. The positions of PMMoV RNA and RNA species derived from partially denatured input dsRNA are indicated. Ethidium bromide staining of 25S rRNA was used as a loading control for the RNA blots (bottom panels).

FIG. 3

Interference of dsRNA with the infection of different plant viruses. (A) dsRNA-mediated interference with TEV. Northern blot analysis of total RNA extracted from inoculated (lanes 1 to 4) or systemic (lanes 5 to 8) leaves of N. tabacum plants challenged with TEV alone or with TEV plus TEV-HC dsRNA at 6 dpi. Similar amounts (5 μg) of each RNA sample were fractionated by 1% agarose gel electrophoresis, and the filter was hybridized with a DIG-labeled RNA probe specific for TEV. The positions of TEV RNA and RNA species derived from partially denatured input TEV-HC dsRNA are indicated. Differences in stability between different RNA samples could probably account for variations in the intensity of the dsRNA bands. Ethidium bromide staining of 25S rRNA was used as a loading control for the RNA gel blot (bottom panel). (B) dsRNA-mediated interference with AMV. Northern blot analysis of total RNA extracted at 6 dpi from inoculated (lanes 1, 3, and 4) or systemic (lanes 6 and 7) leaves of N. benthamiana plants challenged with AMV RNAs 1, 2, and 3 alone (−) or with this mixture plus AMV-3 dsRNA. M, RNA extracted from a mock-inoculated plant. In vitro-transcribed AMV RNAs 3 and 4 (lane 2) and AMV-3 dsRNA (lane 5) used in the inoculum were run for comparison. Similar amounts (1 μg) of RNA samples were fractionated by 1.2% agarose gel electrophoresis, and the filter was hybridized with a DIG-labeled RNA probe specific for AMV RNA3. The positions of AMV RNAs 3 and 4 and input AMV-3 dsRNA are indicated. Ethidium bromide staining of 25S rRNA was used as a loading control for the RNA blot (bottom panel).

FIG. 4

Dose-dependent interference with PMMoV infection by dsRNA. Northern blot analysis of total RNA extracted from inoculated (lanes 1 to 6) or uppermost systemic (lanes 7 to 12) leaves of N. benthamiana at 6 and 15 dpi, respectively. Plants were inoculated with PMMoV (5 μg/ml) alone (−) or with PMMoV plus a series of log dilutions (10⁰ to 10⁻⁴) of the 54-kDa-protein dsRNA, as indicated. Similar amounts (1 μg) of RNA samples were fractionated by 1% agarose gel electrophoresis, and the filter was hybridized with a DIG-labeled 54-kDa-protein RNA probe. The positions of PMMoV RNA and RNA species derived from partially denatured, input 54-kDa-protein dsRNA are indicated. The minor, low-molecular-weight bands in lanes 4 and 6 are probably degradation products of genomic RNA. Ethidium bromide staining of 25S rRNA was used as a loading control for the RNA blot (bottom panel).

FIG. 5

Agrobacterium-mediated transient expression of 54-kDa-protein dsRNA interferes with PMMoV infection. Leaves of N. benthamiana plants were initially infiltrated as indicated with A. tumefaciens cultures carrying either the sense (S) or the antisense (As) 54-kDa-protein RNA-expressing vector, or a mixture of both cultures. After 4 days, the agroinfiltrated leaves of these plants were challenge inoculated with PMMoV or were mock inoculated (M). After another 15 days, accumulation of PMMoV RNA was assessed on inoculated (lanes 1 to 7) and upper (lanes 8 to 14) leaves of two plants per treatment by Northern blot analysis. Similar amounts (1 μg) of RNA samples were fractionated by 1% agarose gel electrophoresis, and the filter was hybridized with a DIG-labeled 54-kDa-protein RNA probe. A faint degradation product of genomic RNA is observed in RNA extracts containing PMMoV RNA. Ethidium bromide staining of 25S rRNA was used as a loading control for the RNA blot (bottom panel).