Suppression of RNA silencing by a geminivirus nuclear protein, AC2, correlates with transactivation of host genes

Abstract

Bipartite geminiviruses encode a small protein, AC2, that functions as a transactivator of viral transcription and a suppressor of RNA silencing. A relationship between these two functions had not been investigated before. We characterized both of these functions for AC2 from Mungbean yellow mosaic virus-Vigna (MYMV). When transiently expressed in plant protoplasts, MYMV AC2 strongly transactivated the viral promoter; AC2 was detected in the nucleus, and a split nuclear localization signal (NLS) was mapped. In a model Nicotiana benthamiana plant, in which silencing can be triggered biolistically, AC2 reduced local silencing and prevented its systemic spread. Mutations in the AC2 NLS or Zn finger or deletion of its activator domain abolished both these effects, suggesting that suppression of silencing by AC2 requires transactivation of host suppressor(s). In line with this, in Arabidopsis protoplasts, MYMV AC2 or its homologue from African cassava mosaic geminivirus coactivated >30 components of the plant transcriptome, as detected with Affymetrix ATH1 GeneChips. Several corresponding promoters cloned from Arabidopsis were strongly induced by both AC2 proteins. These results suggest that silencing suppression and transcription activation by AC2 are functionally connected and that some of the AC2-inducible host genes discovered here may code for components of an endogenous network that controls silencing.

FIG. 1.

MYMV AC2 is a transactivator of viral transcription. Transient expression in N. plumbaginifolia protoplasts of a reporter gene (CAT) driven by the MYMV DNA A rightward (AV2) promoter (scheme of the construct pAC1AV2CAT on top left) and the control CaMV 35S promoter (p35SCAT), in the presence or absence of the AC2-expressing construct (top right), was measured 20 to 24 h posttransfection. Relative expression values indicated are the means for six independent experiments (standard error did not exceed 25% of the mean values; indicated by error bars). The exclamation mark represents CaMV 35S terminator sequences.

FIG. 2.

MYMV AC2 is a nuclear protein with a split NLS. Subcellular localization of GFP-fused wild type AC2 protein (WT) and its mutant variants (AD⁻, ZF⁻, NLS1⁻, and NLS2⁻) following transient expression driven by the 35S promoter with double enhancer (e35S; ChS, portion of chalcone synthase gene; see the text) in N. plumbaginifolia protoplasts was detected in individual plant cells (stained with DAPI) by using confocal fluorescence microscopy. Filtered fluorescence images of GFP-AC2 (left) and DAPI-stained nuclear DNA (center) for each cell were merged (right): if GFP-AC2 fusion (green) is localized to the nucleus (dark blue), the latter appears light blue in the merge image (WT, AD⁻, ZF⁻, and, less pronouncedly, NLS2⁻).

FIG. 3.

MYMV AC2 is a suppressor of RNA silencing. (A) GFP silencing in N. benthamiana line 16c seedlings was triggered by biolistic delivery of a GFP plasmid and visualized under UV light after 6 days (local silencing, left image), 2 weeks (systemic silencing, middle image) and 2 months (total silencing, right image). (B) Codelivery of the wild-type MYMV AC2 reduced local silencing (left) and totally suppressed systemic silencing (middle and right images). (C) MYMV AC2 mutants AD⁻, ZF⁻, and NLS1⁻ did not exhibit any antisilencing effect. (D) RNA blot hybridization performed as described by Klahre et al. (26). Accumulation of GFP siRNAs (thick arrow) in the presence of MYMV AC2 (lanes 4 and 7; note a fourfold-higher loading of total RNA in lane 7) was lower than in the absence of AC2 (lane 5). No GFP siRNAs could be visualized in “green” tissue in the absence of the silencing trigger (lanes 3 and 6). Besides the siRNAs, the antisense GFP riboprobe hybridized specifically to a synthetic 21-nt sense GFP RNA (lane 1) but not to a synthetic 21-nt antisense GFP RNA (lane 2), both described by Klahre et al. (26). A similar pattern of GFP siRNAs in the plant tissue samples was also detected with a sense GFP probe (data not shown).

FIG. 4.

Response of the Arabidopsis transcriptome to individual geminivirus proteins. (A) MYMV proteins AC2 (or its mutants AD⁻ and ZF⁻) and AC1 or ACMV AC2 was expressed individually in Arabidopsis protoplasts, and RNA profiling was performed by using Affymetrix ATH1 GeneChips. (B) The host genes with increased (top panels) or decreased (bottom panels) RNA levels are shown with respect of their physical location on the five chromosomes. (C) Venn diagram showing overlap between the lists of the genes increased >2-fold in response to the respective viral proteins.