Geminivirus AL2 and L2 proteins suppress transcriptional gene silencing and cause genome-wide reductions in cytosine methylation

Abstract

Geminiviruses replicate single-stranded DNA genomes through double-stranded intermediates that associate with cellular histone proteins. Unlike RNA viruses, they are subject to RNA-directed methylation pathways that target viral chromatin and likely lead to transcriptional gene silencing (TGS). Here we present evidence that the related geminivirus proteins AL2 and L2 are able to suppress this aspect of host defense. AL2 and L2 interact with and inactivate adenosine kinase (ADK), which is required for efficient production of S-adenosyl methionine, an essential methyltransferase cofactor. We demonstrate that the viral proteins can reverse TGS of a green fluorescent protein (GFP) transgene in Nicotiana benthamiana when overexpressed from a Potato virus X vector and that reversal of TGS by geminiviruses requires L2 function. We also show that AL2 and L2 cause ectopic expression of endogenous Arabidopsis thaliana loci silenced by methylation in a manner that correlates with ADK inhibition. However, at one exceptional locus, ADK inhibition was insufficient and TGS reversal required the transcriptional activation domain of AL2. Using restriction-sensitive PCR and bisulfite sequencing, we showed that AL2-mediated TGS suppression is accompanied by reduced cytosine methylation. Finally, using a methylation-sensitive single-nucleotide extension assay, we showed that transgenic expression of AL2 or L2 causes global reduction in cytosine methylation. Our results provide further evidence that viral chromatin methylation is an important host defense and allow us to propose that as a countermeasure, geminivirus proteins reverse TGS by nonspecifically inhibiting cellular transmethylation reactions. To our knowledge, this is the first report that viral proteins can inhibit TGS.

FIG. 1.

Overexpression of AL2 and L2, as well as geminivirus infection, reverses TGS of a GFP transgene. (A) Transgenic N. benthamiana plants containing a transcriptionally silenced 35S-GFP transgene (line 16-TGS) were inoculated with PVX vector or PVX expressing the indicated viral protein. Plants were photographed under UV light 2 weeks postinoculation. Uniform dark red color resulting from chlorophyll autofluorescence in the absence of GFP is evident in the PVX-inoculated plant. Release of TGS is indicated by the presence of GFP (yellow-green color) in veins and mesophyll of upper leaves following inoculation with PVX::AL2, AL2_1-114, AL2-C33A, and L2. Results shown are representative of at least three independent trials with 4 to 8 plants per treatment. (B) As described for panel A except that 16-TGS plants were inoculated with VIGS TRV vector or TRV expressing sequences derived from the indicated endogenous genes in order to reduce their expression. Plants were photographed under UV light 4 weeks postinoculation. (C) Line 16-TGS plants were inoculated with PVX (negative control), PVX::AL2 (positive control), CaLCuV, BCTV, or BCTV L2-2 mutant (L2⁻) virus. Plants were photographed under UV light three weeks postinoculation. Results shown are representative of two independent trials with 16 plants per treatment. (D) Northern blot of RNA from leaves of 16-TGS plants inoculated as indicated. The ³²P-labeled probe was specific for GFP mRNA. The 18S rRNA loading controls were visualized by staining the gel with ethidium bromide. (E) Nuclear run-on analysis indicating GFP transcription in line 16C and transcriptional silencing in derivative line 16-TGS. Rubisco served as a control.

FIG. 2.

AL2, AL2_1-114, and L2 cause overexpression of methylated, TGS-silenced loci. Arabidopsis plants containing the indicated transgenes under the control of a dex-inducible promoter were treated with dex (+) or water (−), and expression from the endogenous silenced loci was evaluated by sqRT-PCR. Actin served as a control. The data are representative of results obtained in at least two experiments using two independent lines for each transgene.

FIG. 3.

dsADK causes ectopic expression of TGS-silenced loci. (A) Arabidopsis plants containing a dex-inducible transgene (dsADK) designed to express ADKds RNA were treated with dex or water, and ADK activity was evaluated. The histogram shows relative ADK activity in extracts of plants from two independent lines (dsADK-L4 and dsADK-L5) following dex or water (mock) treatment. Mock extracts were obtained from a dsADK-L4 plant. (B) Expression of the indicated endogenous loci in extracts from dsADK plants treated with dex (+) or water (−) was assessed by sqRT-PCR. 18S rRNA served as control. Lane 1, mock; lane 2, dsADK-L4; lane 3, dsADK-L5.

FIG. 4.

Restriction-sensitive PCR shows reduced cytosine methylation in the Athila and AtSN1 loci following expression of AL2. Arabidopsis plants containing a dex-inducible AL2 transgene (one or two independent lines) were treated with dex (+) or water (−). DNA extracts were incubated with the indicated methylation-sensitive restriction enzymes prior to genomic PCR with primers flanking the restriction site. Destruction of template due to reduced methylation is expected to result in a smaller amount of PCR product. 18S rDNA served as control.

FIG. 5.

Bisulfite sequencing reveals reduced cytosine methylation in the AtSN1 locus following expression of AL2. DNA was obtained from dex-inducible AL2 transgenic Arabidopsis plants after they were sprayed with dex or water (mock). Following bisulfite treatment, the AtSN1 locus was amplified by PCR and cloned. Six clones each were sequenced from dex- or water-treated plants. (A) Summary of bisulfite sequencing data. Red represents CG sites, green CNG sites, and blue CHH sites. Height of bars is proportional to the number of clones with a methylated cytosine, ranging from 0 to 6. The 156-nt sequence is divided into three parts, with data from water treatment shown above data from dex treatment. (B) The histogram represents the proportions of cytosine residues methylated in different sequence contexts. m5C, 5-methylcytosine.

FIG. 6.

Transgenic expression of AL2 and L2 reduces genome-wide cytosine methylation. The histograms illustrate relative incorporation of dCTP observed in methylation-sensitive extension assays. DNA was obtained from dex- or water (mock)-treated Arabidopsis plants containing dex-inducible AL2 or L2 transgenes, digested to completion with MspI, and incubated with [³²P]dCTP and Taq polymerase to allow single-nucleotide extension (see text). Increased incorporation reflects enhanced MspI cleavage due to reduced methylation. Assays were performed with DNA from three individual plants from each of two independent AL2 transgenic lines (AL2-L6 and AL2-L7) (A) or three individual plants from each of two independent L2 transgenic lines (L2-L6 and L2-L8) (B). Mock treatment assays contained DNA from three or four plants from the same transgenic lines. Asterisks indicate significant differences between dex- and mock-treated samples at the 95% (*) or 99% (**) confidence level, as determined by Student's t test.