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. 2008 Sep;82(18):8997-9007.
doi: 10.1128/JVI.00719-08. Epub 2008 Jul 2.

Viral genome methylation as an epigenetic defense against geminiviruses

Priya Raja  1 , Bradley C Sanville, R Cody Buchmann, David M Bisaro
Affiliations

Viral genome methylation as an epigenetic defense against geminiviruses

Priya Raja et al. J Virol. 2008 Sep.

Abstract

Geminiviruses encapsidate single-stranded DNA genomes that replicate in plant cell nuclei through double-stranded DNA intermediates that associate with cellular histone proteins to form minichromosomes. Like most plant viruses, geminiviruses are targeted by RNA silencing and encode suppressor proteins such as AL2 and L2 to counter this defense. These related proteins can suppress silencing by multiple mechanisms, one of which involves interacting with and inhibiting adenosine kinase (ADK), a cellular enzyme associated with the methyl cycle that generates S-adenosyl-methionine, an essential methyltransferase cofactor. Thus, we hypothesized that the viral genome is targeted by small-RNA-directed methylation. Here, we show that Arabidopsis plants with mutations in genes encoding cytosine or histone H3 lysine 9 (H3K9) methyltransferases, RNA-directed methylation pathway components, or ADK are hypersensitive to geminivirus infection. We also demonstrate that viral DNA and associated histone H3 are methylated in infected plants and that cytosine methylation levels are significantly reduced in viral DNA isolated from methylation-deficient mutants. Finally, we demonstrate that Beet curly top virus L2- mutant DNA present in tissues that have recovered from infection is hypermethylated and that host recovery requires AGO4, a component of the RNA-directed methylation pathway. We propose that plants use chromatin methylation as a defense against DNA viruses, which geminiviruses counter by inhibiting global methylation. In addition, our results establish that geminiviruses can be useful models for genome methylation in plants and suggest that there are redundant pathways leading to cytosine methylation.

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Figures

FIG. 1.
FIG. 1.
The methylation pathway in plants. A putative pathway for RNA-directed DNA methylation in Arabidopsis is illustrated. Genomic and viral genome targets may be transcribed by an RNA polymerase IVa complex (Pol IVa; containing NRPD1A and NRPD2). Resulting single-stranded RNA (ssRNA) is converted to dsRNA by complexes containing RDR2. The 24-nt siRNAs processed from dsRNA by DCL3 are loaded into complexes containing AGO4, which subsequently associates with Pol IVb (containing NRPD1B and NRPD2). The AGO4-associated siRNAs target the complex to homologous DNA sequences, where cytosine methyltransferases (e.g., DRM1/2) are recruited. Methylation also involves the SWI-SNF chromatin remodeling activities DRD1 and DDM1. Cytosine methyltransferases CMT3 and MET1 are primarily involved in methylation maintenance at CNG and CG sites, respectively. CNG methylation by CMT3 is also linked to H3K9 methylation carried out by KYP2.
FIG. 2.
FIG. 2.
Methylation-deficient mutant plants are hypersusceptible to geminivirus infection. Photographs are illustrative of geminivirus disease symptoms on wild-type and selected mutant plants. Plants were mock inoculated or inoculated with virus within 5 days of bolting, and symptoms were observed after 10 to 14 days for CaLCuV and 14 to 21 days for BCTV. Methylation-deficient mutants showed greater stunting and increased inflorescence deformation in response to geminivirus infection than wild-type (WT) plants of the same ecotype. (A) Arabidopsis Ler-0 and cmt3 mutant plants inoculated with BCTV. (B) Col-0 and adk1 mutant plants inoculated with CaLCuV. (C) Typical inflorescence structures of BCTV-infected kyp2, cmt3, and ago4 mutants in the Ler-0 background. (D) Inflorescence structures of CaLCuV-infected adk2, nrpd2a, and met1 mutants in the Col-0 background. (E) Inflorescence structures of BCTV-infected ddm1, rdr2, rdr6, dcl2, dcl3, dcl4, and dcl2 dcl3 dcl4 mutants in the Col-0 background. (F) Inflorescence structures of BCTV-infected drm1 drm2 and mmt mutants in the Ws-2 background.
FIG. 3.
FIG. 3.
The IRs of CaLCuV and BCTV are methylated in infected plants, and methylation levels are reduced in methylation-deficient mutants. Viral DNA isolated from floral tissues of infected wild-type plants or infected mutant plants was treated with bisulfite, and the IR was amplified by PCR. Amplified fragments were cloned, and 12 to 18 (CaLCuV) or 7 to 10 (BCTV) independent clones representing the encapsidated, viral strand were sequenced for each treatment. (A) The sequence of the CaLCuV IR, summarizing methylation detected in viral DNA isolated from wild-type plants of the ecotypes Ler-0, Ws-2, and Col-0, is shown. Methylated cytosines are indicated in red. Cytosines that were unmethylated in all three ecotypes are indicated in green. Cytosines methylated in 5 to 10% of the clones are additionally noted by a dot, and more frequently methylated cytosines (>10 to 20% of the clones) are marked by an asterisk. Sites that show reduced methylation (zero to one clone) in the mutants are underlined. The locations of the conserved geminivirus hairpin and the putative, imperfectly repeated AL1 binding sites (AGGGGAG/AGGAGAG) are shown. The locations of start codons for the divergent AL1 and coat protein genes are also indicated. (B) The histograms represent the percentages of cytosine sites methylated in different sequence contexts in CaLCuV IR DNA isolated from N. benthamiana (N. benth.) and Arabidopsis wild-type (Ler-0, Ws-2, and Col-0) plants and the indicated mutant plants (adk2, ago4, cmt3, drm1 drm2, and kyp2). Mutants are compared to the appropriate wild-type background. (C) Histograms represent the percentages of total cytosine residues methylated in different sequence contexts in BCTV IR DNA isolated from N. benthamiana and Arabidopsis wild-type Ler-0 plants, and ago4, kyp2, and cmt3 mutants.
FIG. 4.
FIG. 4.
ChIP analysis of the CaLCuV IR. Tissue from CaLCuV-infected N. benthamiana (A) or Arabidopsis (B) plants was cross-linked with formaldehyde and subjected to ChIP using antibodies specific for the indicated histone H3 modifications. PCR was performed to amplify a ∼400-bp fragment spanning the ∼300-bp IR. The H3 acetyl polyclonal antibody was raised against a peptide acetylated at lysines 9 and 14. Controls included actin and transposons Tnt and Ta3. Additional control experiments in which plasmid DNA or plasmid DNA containing the CaLCuV genome was added to healthy plant extracts yielded similar signals with actin primers, but no signals were detected with the CaLCuV IR primers (data not shown). AB, antibody.
FIG. 5.
FIG. 5.
Arabidopsis ago4 mutants do not recover after infection with BCTV L2− mutant virus. (A) The photographs show secondary (2°) tissue of a wild-type (Ler-0) and ago4 mutant plant infected with BCTV L2-1. Note recovery (absence of symptoms) in wild-type plants and severe disease symptoms in the ago4 mutant. (B) Southern blot hybridization analysis of BCTV L2-1 and L2-2 DNA in primary (1°) and secondary (2°) infected tissues of wild-type and ago4 plants. DNA extracts were prepared from pools of at least six plants and digested with ScaI to linearize the circular viral genome and cleave host genomic 18S ribosomal DNA (rDNA) repeats. Blots were incubated with a BCTV-specific probe and later with a probe specific for 18S rDNA to provide a loading control. Viral single-stranded DNA (ss) and linear dsDNA (ds) are indicated. Note reduced BCTV L2-1 and L2-2 DNA levels in recovered, wild-type secondary tissue. (C) Methylation of the BCTV and BCTV L2-1 and L2-2 IRs. The histograms represent the percentages of total cytosine residues methylated in different sequence contexts in viral DNA obtained from wild-type and ago4 mutants. Note the greatly increased methylation observed in BCTV L2-1 and L2-2 DNA obtained from wild-type secondary tissue exhibiting recovery from infection. WT, wild type.
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References

    1. Akbergenov, R., A. Si-Ammour, T. Blevins, I. Amin, C. Kutter, H. Vanderschuren, P. Zhang, W. Gruissem, F. Meins, Jr., T. Hohn, and M. M. Pooggin. 2006. Molecular characterization of geminivirus-derived small RNAs in different plant species. Nucleic Acids Res. 34462-471. - PMC - PubMed
    1. Baulcombe, D. C. 2004. RNA silencing in plants. Nature 431356-363. - PubMed
    1. Bender, J. 2004. DNA methylation and epigenetics. Annu. Rev. Plant Biol. 5541-68. - PubMed
    1. Bian, X.-Y., M. S. Rasheed, M. J. Seemanpillai, and M. A. Rezaian. 2006. Analysis of silencing escape of Tomato leaf curl virus: an evaluation of the role of DNA methylation. Mol. Plant-Microbe Interact. 19614-624. - PubMed
    1. Bisaro, D. M. 2006. Silencing suppression by geminivirus proteins. Virology 344158-168. - PubMed

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