Negative-strand tospoviruses and tenuiviruses carry a gene for a suppressor of gene silencing at analogous genomic positions

Abstract

Posttranscriptional silencing of a green fluorescent protein (GFP) transgene in Nicotiana benthamiana plants was suppressed when these plants were infected with Tomato spotted wilt virus (TSWV), a plant-infecting member of the BUNYAVIRIDAE: Infection with TSWV resulted in complete reactivation of GFP expression, similar to the case for Potato virus Y, but distinct from that for Cucumber mosaic virus, two viruses known to carry genes encoding silencing suppressor proteins. Agrobacterium-based leaf injections with individual TSWV genes identified the NS(S) gene to be responsible for the RNA silencing-suppressing activity displayed by this virus. The absence of short interfering RNAs in NS(S)-expressing leaf sectors suggests that the tospoviral NS(S) protein interferes with the intrinsic RNA silencing present in plants. Suppression of RNA silencing was also observed when the NS3 protein of the Rice hoja blanca tenuivirus, a nonenveloped negative-strand virus, was expressed. These results indicate that plant-infecting negative-strand RNA viruses carry a gene for a suppressor of RNA silencing.

FIG. 1.

Schematic representation of the coding strategies of the TSWV and RHBV genomes. The dashed line surrounding the tenuivirus RHBV indicates that no membranous particles have been found for these viruses, in contrast to the case for tospoviruses and other members of the Bunyaviridae. Viruses of both genera have a fully negative-stranded large RNA (L RNA and RNA 1, respectively), encoding the viral RdRP. The nucleoprotein is encoded on the virus cRNA strand of the third-largest segment (S RNA or RNA 3), in which the NS_S and NS3 proteins are encoded on the viral RNA strand. The remaining RNA segments of both viruses are all ambisense.

FIG. 2.

Virus inoculation of transgenic N. benthamiana plants containing a silenced GFP gene. (A) Suppression of RNA silencing by TSWV. The plant on the left is a nontransgenic plant infected with TSWV to show that the infection does not cause autofluorescence. The plant on the right is a noninfected, GFP-silenced control plant. The photograph was taken without a filter. (B) Recovery of GFP expression in GFP-silenced plants by infection with TSWV or PVY at 10 days postinoculation. (C) Suppression of GFP expression in older leaves. Photographs were taken at 3 weeks postinoculation. In panels B and C, a Kodak Wratten no. 58 filter was used.

FIG. 3.

Agrobacterium infiltration experiments with different TSWV genes in GFP-silenced N. benthamiana plants. Agrobacterium strains harboring TSWV genes were coinfiltrated with GFP. Only NS_S suppresses the silencing of GFP (panel D).

FIG. 4.

RNA silencing suppression activity displayed by HC-Pro (PVY), NS_S (TSWV), and NS3 (RHBV). The picture was taken with a yellow filter at 6 days after infiltration.

FIG. 5.

(A) GPF imaging of Agrobacterium-infiltrated leaves from nontransgenic plants. GFP expression was visualized by UV light in leaves coinfiltrated with GFP and an empty vector, CABMV HC-Pro, TSWV NS_S, RHBV NS3, or CMV 2b. (B) Total protein was extracted from corresponding infiltrated leaf sectors. Western blotting was performed with anti-GFP antibodies. (C) Northern blot analysis of total mRNA extracted from the infiltrated leaf parts. Ethidium bromide staining of the same gel shows the 25S rRNA as a loading control.

FIG. 6.

Analysis of the GFP siRNAs by RNase A/T₁ protection assay. Small RNA-enriched samples were extracted from Agrobacterium-infiltrated sectors of GFP-silenced leaves. Noninfiltrated nontransgenic plants were used as a control. All other leaves were coinfiltrated with GFP and (putative) silencing suppressor constructs. Twenty micrograms of RNA was hybridized to sense GFP RNA transcripts and subsequently treated with the RNases A and T₁. Sizes of RNA oligonucleotides are indicated on the right in bases.