Viral virulence protein suppresses RNA silencing-mediated defense but upregulates the role of microrna in host gene expression

Abstract

Small interfering RNAs (siRNAs) and microRNAs (miRNAs) are processed by the ribonuclease Dicer from distinct precursors, double-stranded RNA (dsRNA) and hairpin RNAs, respectively, although either may guide RNA silencing via a similar complex. The siRNA pathway is antiviral, whereas an emerging role for miRNAs is in the control of development. Here, we describe a virulence factor encoded by turnip yellow mosaic virus, p69, which suppresses the siRNA pathway but promotes the miRNA pathway in Arabidopsis thaliana. p69 suppression of the siRNA pathway is upstream of dsRNA and is as effective as genetic mutations in A. thaliana genes involved in dsRNA production. Possibly as a consequence of p69 suppression, p69-expressing plants contained elevated levels of a Dicer mRNA and of miRNAs as well as a correspondingly enhanced miRNA-guided cleavage of two host mRNAs. Because p69-expressing plants exhibited disease-like symptoms in the absence of viral infection, our findings suggest a novel mechanism for viral virulence by promoting the miRNA-guided inhibition of host gene expression.

Figure 1.

p69 Is a PTGS Suppressor in A. thaliana. (A) TYMV genome organization. (B) to (D) Suppression of the GUS RNA silencing in line L1 by TYMV 2 weeks after infection (B), by a 35S-P69 transgene (C), or a TYMV amplicon transgene (D). Photos of the same plants before GUS staining are also shown in the bottom panels in (C) and (D).

Figure 2.

p69-Conferred Virulence in A. thaliana. Transgenic line P69c (left) expressing P69 causes pleiotropic developmental defects that resemble the phenotype displayed by a plant infected systemically with TYMV (right).

Figure 3.

Molecular Characterization of P69 Suppression of PTGS Induced by a Sense RNA Transgene in A. thaliana. The detection of the GUS mRNA (A), GUS siRNAs (B), and GUS DNA methylation (C) in sgs2(L1), L1xC24, L1xP69c, and L1xP69d seedlings. In contrast with HpaII, MspI cleavage is not blocked to methylation at sites with overlapping CG. Equal loading and completion of DNA digestion was demonstrated by rehybridization of the filter for SPL3 (bottom panel of [C]), whereas equal loading of small RNAs was shown by reprobing for 5S rRNA (B). The positions of 21- and 26-nucleotide RNA markers are indicated (B).

Figure 4.

Molecular Characterization of p69 Suppression of PTGS Induced by a Virus-Derived Amplicon Transgene. (A) Silencing suppression in amplicon lines infected with TYMV (lanes 4 to 5). Samples from mock-inoculated lines were included as controls (lanes 1 to 3). PVX:GFP genomic (g) and subgenomic (sg) RNAs as well as GFP mRNA are indicated. (B) Silencing suppression by the P69 transgene introduced into lines A and GxA by genetic crosses with the maternal parent listed first (this is used throughout the text). Except for G and sde1(GxA), all plants analyzed were hemizygous F1 plants. Total RNA loaded was 5 μg in lanes 1 to 3 and 3 μg in lanes 4 to 13 and equal loading was visualized by methylene blue staining of rRNA. (C) Detection of siRNAs by a GFP-specific probe in hemizygous F1 amplicon plants crossed with P69c, P69d, or C24. A similar pattern of siRNA accumulation was detected using a PVX-specific probe. (D) Detection of the GFP DNA methylation. Note that HaeIII cleavage is not blocked to methylation at sites with overlapping CG. The positions of DNA standards (in base pairs) are shown at the right. (E) Detection of TYMV high and low molecular weight RNAs in either the TYMV-based amplicon plants or wild-type plants infected TYMV. The filters were probed with labeled DNA and RNA sequences corresponding to nucleotides 5591 to 6260, respectively, of the TYMV genome. The amplicon transgene contained an insertion of nonviral sequence (1.6 kb) upstream of the viral CP gene and thus gave rise to a recombinant viral genomic RNA longer than the wild-type TYMV genomic RNA, although the size of the CP subgenomic RNA remained unaltered.

Figure 5.

p69 Does Not Suppress the IR-RNA–Induced PTGS Targeting of PDS. (A) to (C) Genetic segregation of the photobleaching RNA interference phenotype of the IRPDS transgene after crossing an IRPDS heterozygote (IRPDS/−, in Col-0 background) with either C-24 or a P69c homozygote (in the C-24 background). A representative plate is shown for each except for P69cxIRPDS, which did not show difference with or without hygromycin selection. P69c plants appear yellow in contrast with the photobleached plants (C). (D) Detection of P69 mRNA and PDS mRNA/siRNAs. For comparison to the level of PDS mRNA in the C24xCol-0 genetic background with and without P69, RNA samples from the progeny of C24xCol-0 (lane 3) and P69cxCol-0 (lane 1) were included as controls. Equal loading was monitored for mRNA by rRNA staining and for small RNAs by reprobing for 5S rRNA.

Figure 6.

Reduced Accumulation of Two miRNA-Targeted mRNAs in p69-Expressing Plants. (A) Detection of full-length SCL6-IV mRNA and its 3′ cleavage product by miR171 in wild-type C-24 and P69c plants. (B) Accumulation of SPL3 mRNA in P69c and P69d plants as well as in C-24 plants before (Wt) and after systemic TYMV infection (V). (C) Determination of the miR156 cleavage sites within SPL3 mRNA. The 5′-terminal portion of the predicted 3′ cleavage product for SPL3 mRNA as well as SPL2 and CUC2 mRNAs (Kasschau et al., 2003; used here as controls) was amplified by 5′RACE (top panel), and the dominant products were recovered, cloned, and sequenced. The arrows indicate the positions of two cleavage sites inferred (bottom panel). The number of the 5′RACE clones that correspond to each site is indicated.

Figure 7.

Enhanced Accumulation of Seven miRNAs and mRNAs for DCL1 and SDE1/SGS2 in p69-Expressing Plants. (A) and (B) Detection of miRNAs in P69c and wild-type C-24 plants as described in Methods. We noted that the level of miR162 was much lower in wild-type ecotype C-24 than in either Landsberg erecta (Ler) or Col-0. (C) Detection of DCL1 and AGO1 mRNAs by RNA gel blot hybridizations. Two micrograms of poly(A)⁺ mRNAs from P69c and C-24 plants were loaded in each lane, and the filter was probed sequentially for the mRNA of AGO1, DCL1, and β-TUBULIN-1 (as a loading control). The full-length DCL1 and AGO1 mRNAs are 6.2 and 3.5 kb, respectively, as indicated. (D) Real-time PCR analyses of the mRNA accumulation for AGO1, DCL1, and β-TUBULIN-1 in P69c and C-24 plants.