Virus-encoded suppressor of posttranscriptional gene silencing targets a maintenance step in the silencing pathway

Abstract

Certain plant viruses encode suppressors of posttranscriptional gene silencing (PTGS), an adaptive antiviral defense response that limits virus replication and spread. The tobacco etch potyvirus protein, helper component-proteinase (HC-Pro), suppresses PTGS of silenced transgenes. The effect of HC-Pro on different steps of the silencing pathway was analyzed by using both transient Agrobacterium tumefaciens-based delivery and transgenic systems. HC-Pro inactivated PTGS in plants containing a preexisting silenced beta-glucuronidase (GUS) transgene. PTGS in this system was associated with both small RNA molecules (21-26 nt) corresponding to the 3' proximal region of the transcribed GUS sequence and cytosine methylation of specific sites near the 3' end of the GUS transgene. Introduction of HC-Pro into these plants resulted in loss of PTGS, loss of small RNAs, and partial loss of methylation. These results suggest that HC-Pro targets a PTGS maintenance (as opposed to an initiation or signaling) component at a point that affects accumulation of small RNAs and methylation of genomic DNA.

Figure 1

Diagram of constructs and Agrobacterium injection strategy. (A) Key portion of the construct used to produce transgenic plants containing a posttranscriptionally silenced GUS sequence. The GUS coding sequence was interrupted by a stop codon/frameshift mutation after codon four (19). The construct, therefore, contains a nontranslatable (nt) GUS sequence and directs no GUS activity in transgenic plants. (B) Key portions of the plasmids used for transient expression in Agrobacterium infiltration assays. The GUS + HC-Pro plasmid contains two independent expression cassettes. (C) Agrobacterium infiltration strategy. Two zones in each leaf were infiltrated with combinations of Agrobacterium cultures containing empty pSLJ755I5 vector, single-cassette GUS plasmid, or dual-cassette GUS + HC-Pro plasmid. Leaves were detached at 4 days after infiltration and subjected to GUS activity assay or immunoblot analysis.

Figure 2

Suppression of PTGS by transient Agrobacterium-mediated delivery of HC-Pro. GUS encoded by single-GUS or dual-GUS + HC-Pro cassettes was detected by histochemical assay in leaf tissue at 4 days after infiltration. (A) Control series of Agrobacterium injection assays with nontransgenic plants (line 13). (B) Dependence of GUS activity on delivery of T-DNA by Agrobacterium. Plasmids were introduced into Vir⁺ and Vir⁻ strains of Agrobacterium, followed by injection into nontransgenic plants. (C) Agrobacterium injection assays with the same series shown in A, but with GUS-silenced plants (line 7). Note that GUS activity occurs only in leaves infiltrated with Agrobacterium containing the dual GUS + HC-Pro plasmid.

Figure 3

Immunoblot analysis of GUS and HC-Pro after Agrobacterium-mediated delivery into GUS-silenced and nontransgenic plant lines. Normalized, total detergent-soluble protein extracts were prepared from tissue injected with Agrobacterium carrying vector alone or plasmids containing the single-GUS or dual-GUS + HC-Pro expression cassettes. Lanes 1–6, nontransgenic line 13. Lanes 7–12, GUS-silenced line 7. Samples consisted of pools of tissue from four injection zones. Two samples are shown for each treatment. Immunoblot results with anti-HC-Pro (Upper) and anti-GUS sera (Lower) are shown.

Figure 4

Detection of short RNAs in GUS-silenced transgenic plants. Low molecular weight RNA was extracted from leaves of either nontransgenic (NT), GUS-silenced (422 and 407), or GUS-nonsilenced (446) plants. Equal amounts of each RNA sample were subjected to electrophoresis in denaturing 15% polyacrylamide gels, stained with ethidium bromide, blotted to a nylon membrane, and hybridized using various ³²P-labeled GUS DNA fragments as probes. The PTGS status of each plant is indicated above the lanes. (A) Two experiments analyzing small RNAs with a full-length, ³²P-labeled GUS probe. In vitro transcribed GUS RNA was hydrolyzed (OH⁻) and used as a hybridization control. The arrow indicates the position of short RNA. (Right) Ethidium bromide staining of the gel used in experiment 2. DNA oligonucleotides (21, 26, and 32 nt) were used as standards (STD). (B) Analysis of small RNAs with normalized (2 × 10⁶ cpm) ³²P-labeled probes corresponding to different regions of the GUS coding sequence. The positions of the 21 and 26 nt DNA standards are shown at the right.

Figure 5

HC-Pro suppresses accumulation of short RNAs. (A) Blot analysis of GUS mRNA in either nontransgenic (NT), GUS-silenced (407 and 17), or silencing-suppressed (17HC) plants. High-molecular weight RNA was isolated, normalized (10 μg/lane), subjected to electrophoresis, blotted to a nylon membrane, and hybridized by using ³²P-labeled full-length GUS DNA as a probe. The blot was stripped and reprobed with a ³²P-labeled DNA probe specific for rRNA. The positions of both GUS and rRNA mRNAs are indicated. (B) Blot analysis of short RNA. Low-molecular weight RNA was isolated and analyzed as described in the legend for Fig. 4. The results from two independent experiments (Expt.) are shown. The arrow indicates the position of silencing-specific short RNA. For presentation purposes, the data shown in A and B are composite images from noncontiguous lanes from a single blot.

Figure 6

HC-Pro partially suppresses methylation of target DNA. (A) Schematic representation of DNA corresponding to the GUS coding sequence. Positions of HaeIII restriction sites (H1-H5) and sizes (in nucleotides) of the expected digestion products are illustrated. Sites marked by an asterisk contain cytosines in a symmetrical (CpNpG) configuration. Filled circles indicate HaeIII sites that were cytosine methylated in GUS-silenced plants. The right (RB) and left (LB) borders of the GUS transgene are indicated. (B) Blot analysis of genomic DNA in nontransgenic (NT), GUS-silenced transgenic (7 and 17), GUS-nonsilenced transgenic (446), and GUS-silencing suppressed (no. 17HC) plants. Blots were hybridized with a ³²P-labeled probe specific for the GUS gene. The blot was stripped and rehybridized with a ³²P-labeled DNA probe specific for the eIF4E coding sequence.