Two plant viral suppressors of silencing require the ethylene-inducible host transcription factor RAV2 to block RNA silencing

Abstract

RNA silencing is a highly conserved pathway in the network of interconnected defense responses that are activated during viral infection. As a counter-defense, many plant viruses encode proteins that block silencing, often also interfering with endogenous small RNA pathways. However, the mechanism of action of viral suppressors is not well understood and the role of host factors in the process is just beginning to emerge. Here we report that the ethylene-inducible transcription factor RAV2 is required for suppression of RNA silencing by two unrelated plant viral proteins, potyvirus HC-Pro and carmovirus P38. Using a hairpin transgene silencing system, we find that both viral suppressors require RAV2 to block the activity of primary siRNAs, whereas suppression of transitive silencing is RAV2-independent. RAV2 is also required for many HC-Pro-mediated morphological anomalies in transgenic plants, but not for the associated defects in the microRNA pathway. Whole genome tiling microarray experiments demonstrate that expression of genes known to be required for silencing is unchanged in HC-Pro plants, whereas a striking number of genes involved in other biotic and abiotic stress responses are induced, many in a RAV2-dependent manner. Among the genes that require RAV2 for induction by HC-Pro are FRY1 and CML38, genes implicated as endogenous suppressors of silencing. These findings raise the intriguing possibility that HC-Pro-suppression of silencing is not caused by decreased expression of genes that are required for silencing, but instead, by induction of stress and defense responses, some components of which interfere with antiviral silencing. Furthermore, the observation that two unrelated viral suppressors require the activity of the same factor to block silencing suggests that RAV2 represents a control point that can be readily subverted by viruses to block antiviral silencing.

Figure 1. ntRAV Interacts with HC-Pro and Delays the Onset of Sense Transgene Silencing when Over-expressed in Tobacco.

(A) Tobacco ntRAV interacts with TEV HC-Pro in in vitro pulldown experiments. 35S-labelled ntRAV co-purifies with HC-Pro-GST (lane 3), but not with GST (lane 2). Lane 1 shows the amount of input 35S-labelled ntRAV protein used in the pulldown experiments. (B) The accumulation of ntRAV mRNA at 24, 30 and 37 days after germination in whole leaves of wild type (WT) tobacco plants (lanes 1–3), plants heterozygous for the silenced 6b5 GUS transgene (WT X 6b5) (lanes 4–6), and plants heterozygous for the silenced 6b5 GUS transgene and expressing the 35S:ntRAV transgene (lanes 7–9). (C) Histochemical staining of leaves from HC-Pro X 6b5 (left panel), WT X 6b5 (center panel) and 35S:ntRAV X 6b5 leaves (right panel) at 26 days after germination. (D) GUS mRNA levels in the veins of leaves of HC-Pro X 6b5 (lane 1), WT X 6b5 (lane 2) and 35SntRAV X 6b5 plants (lane 3) at 26 days after germination.

Figure 2. In vivo Interaction of RAV2 and TuMV HC-Pro in Arabidopsis.

Proteins isolated from plants expressing either FLAG-tagged RAV2 (Flag-RAV2) alone, TuMV HC-Pro alone or both Flag-RAV2 and TuMV HC-Pro were incubated with anti-FLAG agarose beads. The bound protein was fractionated on acrylamide gels and subjected to western blot analysis using either HC-Pro antiserum (left panel) or RAV2 antiserum (center panel). The far right panel shows the relative input amounts of protein used in the pulldown experiments as determined by Coomassie blue staining.

Figure 3. RAV2 is Required for HC-Pro Suppression of Virus Induced Gene Silencing (VIGS).

(A) Phenotype of plants bombarded with CaLCV vector carrying a portion of the endogenous CH42 gene. VIGS of CH42 results in pronounced yellowing in wild type or rav2 knockout plants (upper left and right panels, respectively). Plants expressing HC-Pro are suppressed for VIGS and therefore remain green (lower left panel); whereas HC-Pro plants in the rav2 knockout background fail to block silencing and display yellowing typical of wild type plants (lower right panel). (B) RNA gel blot analysis of CH42 mRNA, HC-Pro and CH42 siRNA levels in wild type (lanes 1 and 2), rav2 knockout (lanes 3 and 4), HC-Pro plants (lanes 5 and 6) and HC-Pro plants in the rav2 background (lanes 7–9) either uninfected (lanes 1, 3, 5 and 7) or after bombardment with the CH42 VIGS vector (lanes 2, 4, 6, 8 and 9). Ethidium staining of rRNA is shown as the loading control for the high molecular weight blots and the hybridization signal for U6 is shown as the loading control for the small RNA blot. The migration of 24 nt siRNAs is marked by an arrow.

Figure 4. RAV2 is Required for Suppression of Hairpin Transgene Silencing by Two Unrelated Viral Suppressors.

(A) Diagrams showing the structures of the 6b4 and 306 transgene loci. The 6b4 locus is an expressing locus which encodes a functional GUS protein. The 306 locus produces a GUS hairpin RNA that acts in trans to silence the 6b4 locus. The locations of the hybridization probes used in parts B, C and D are indicated. (B and D) The accumulation of RAV2, TCV-P38 and/or TuMV HC-Pro mRNA in plants of the genotypes indicated at the top of the lanes. (C and E) The top panel of each shows the accumulation of 6b4 GUS mRNA in plants of the genotypes indicated at the top of the lanes, and the bottom two panels show the accumulation of primary and secondary siRNAs in the same samples. The size of 21-, 22- and 24-nt marker RNAs are indicated to the left of the small RNA panels and the probes used are indicated to the right of each panel.

Figure 5. RAV2 is Required for Many HC-Pro-associated Morphological Anomalies but not for Defects in MicroRNA Biogenesis.

(A) Flower morphological defects in HC-Pro transgenic plants (top left panel) are rescued in the rav2 knockout background (top middle panel) resulting in flower phenotype indistinguishable from wild type (top right panel). Rosette dwarfing and leaf serration in transgenic plants (bottom left panel) are partially rescued in the rav2 knockout background (bottom middle panel) resulting in a phenotype intermediate between wild type (bottom right panel) and Hc-Pro plants. (B) The accumulation of the indicated miRNAs and miRNA*s was determined from RNA gel blot analysis of low molecular weight RNA from wild type (WT), rav2 knockout plants (rav2), HC-Pro plants (HC) and HC-Pro plants in the rav2 knockout background (rav2, HC). Ethidium bromide (EtBr) staining of the predominant RNA species in the low molecular weight fraction is shown as a loading control.

Figure 6. Tiling Microarray Analysis and RT qPCR Show RAV2-dependent Up-regulation of Silencing-associated Genes by HC-Pro.

(A) The mRNA levels for AGO2 (At1g31280), FRY1 (At5g63980) and CML38 (At1g76650) in rav2 knockout plants (rav2), HC-Pro transgenic plants (HC), HC plants in the rav2 knockout background (rav2/HC) and wild type control plants (WT) were determined by oligo(dT)-primed RT qPCR analysis. Error bars, ±SD. (B) The mRNA levels for the same genes shown in (A) were determined by Arabidopsis whole-genome tiling microarray expression analysis. The top four tracks show the level of these mRNAs in the genotypes indicated to the left of the track. The bottom track indicates the annotated gene models for the three loci. (C) Gene ontology (GO) analysis results for genes that are up-regulated in HC-Pro transgenic plants as compared to wild type plants. The top five over-represented biological processes categories and the associated hypergeometric distribution P-values are shown. (D) GO analysis results for genes that are up-regulated by HC-Pro in a RAV2-dependent manner. The top five over-represented biological processes categories and the associated hypergeometric distribution P-values are shown.