N. benthamiana plants were inoculated with TYLCCNV alone or in combination with TYLCCNB. Samples were prepared by pooling six leaves from six systemically infected plants 21 days post-inoculation. DNA extracts were obtained, and the circular viral genome was divided into six fragments and PCR amplified after bisulfite treatment. Twenty clones from each fragment were sequenced. (A) The TYLCCNV genome is depicted in linear form, beginning within the conserved hairpin located in the intergenic region (IR). The positions of viral genes are shown by arrows. The six fragments used for bisulfite sequencing are indicated (MeA1 to MeA6). (B) The graph shows the total number of methylated cytosines at each position in the TYLCCNV genome, from a total of 20 sequenced clones, in different sequence contexts (CG red, CHH green, CNG blue). (C) As in (B), except DNA was obtained from plants co-inoculated with TYLCCNV + TYLCCNB. (D) The sequence contexts of all cytosines in the ∼2,737 bp TYLCCNV strain Y10 genome are indicated. (E) The histogram shows the percentage of cytosine residues methylated in different sequence contexts in the TYLCCNV genome in plants inoculated with TYLCCNV alone or together with TYLCCNB. Student's t test was performed on methylation values using individual clones as data points. The double asterisks indicate a significant difference between samples with and without TYLCCNB at the 99% confidence interval.

The histograms depict the relative ³²P-dCTP incorporation observed in methylation-sensitive extension assays. DNA was obtained from mock inoculated or infected plants, digested with MspI, and incubated with ³²P-dCTP and Taq polymerase to permit single-nucleotide extension. Increased incorporation due to enhanced MspI cleavage indicates reduced methylation. (A) DNA was obtained from mock-inoculated plants, or plants inoculated with TYLCCNV or TYLCCNV + TYLCCNB. (B) DNA was obtained from mock-inoculated plants, or plants inoculated with PVX or PVX expressing βC1 protein (PVX-βC1). Both (A) and (B) represent data from two independent experiments. In each experiment, assays were performed in duplicate with DNA from three individual plants per treatment. Values represent means +/- SE. Double asterisks indicate significant differences between samples with and without TYLCCNB at the 99% confidence interval, as determined by Student's t test.

(A) Arabidopsis plants from three independent lines containing a dex-inducible βC1 transgene (dex-βC1) were treated with dex (+) or mock-treated (-), and expression of a putative F-box locus known to be methylated and transcriptionally silenced was assessed by sqRT-PCR. Actin served as a control. Mock treated samples were prepared by pooling RNA from the three transgenic lines. Data shown are representative of at least three independent experiments. (B) The histogram illustrates relative incorporation observed in the methylation-sensitive extension assay using MspI. Extracts from two transgenic lines (βC1-L25 and βC1-L31) were tested following dex treatment. Two independent experiments were performed. In each experiment, assays were performed in duplicate with DNA from three individual plants per treatment. Mock treatment assays contained DNA isolated from plants of both transgenic lines. Values represent means +/- SE. Asterisks indicate significant differences between mock and dex-treated samples at the 95% confidence interval, as determined by Student's t test.

(A) Diagram of the assay. In a reversible reaction, S-adenosyl homocysteine (SAH) is hydrolyzed by SAHH to adenosine and homocysteine (Hcy). Conversion of adenosine to labeled adenosine monophosphate (AMP*) by ADK in the presence of labeled γ³²P-ATP drives the reaction in the direction of hydrolysis and allows indirect quantitation of the SAHH reaction. SAH hydrolysis promotes flux through the methyl cycle and SAM production, and this is inhibited by βC1 protein. (B) Autoradiograph of a representative chromatogram showing AMP generated by ADK in reactions containing varying molar ratios of βC1 to SAHH. The positions of labeled AMP product and ATP substrate are indicated. ADK activity (AMP/AMP+ATP) in each reaction was calculated after phosphorimager quantitation of radioactivity in individual spots. GST:SAHH (30∶1) served as a control. (C) Stoichiometry of inhibition. The graph illustrates relative ADK activity, an indirect measure of SAHH activity, with increasing βC1:SAHH molar ratio. Data were obtained from three independent experiments with two independent SAHH preparations. Values represent means +/− SE.

S-adenosyl methionine (SAM) is the methyl donor for most transmethylation reactions. The product, S-adenosyl homocysteine (SAH), inhibits transmethylation by competing with SAM for methyltransferases (MTases). SAH is converted to homocysteine (Hcy) and adenosine by S-adenosyl homocysteine hydrolase (SAHH). Phosphorylation of adenosine by adenosine kinase (ADK) is critical because the SAHH-catalyzed reaction is reversible and the equilibrium lies in the direction of SAH synthesis. By removing adenosine, ADK promotes flux through the cycle and SAM production, and minimizes competitive inhibition of methyltransferase reactions by SAH. Thus, ADK inactivation by geminivirus AC2/AL2 and C2/L2 proteins globally interferes with methylation. Data presented in this report suggests that TYLCCNV AC2/AL2 may be deficient for ADK inhibition. The C2/L2 protein has also been shown to stabilize SAM decarboxylase (SAMDC), which causes decarboxylated SAM (dcSAM) levels to rise. dcSAM is a competitive inhibitor of SAM. Whether AC2/AL2 proteins also promote increases in dcSAM has yet to be tested. Data in this report indicates that the betasatellite encoded βC1 protein directly antagonizes the methyl cycle by inhibiting SAHH. Note that this diagram lists only DNA and histones as methyltransferase substrates, although any type of transmethylation reaction may require SAM as a cofactor. THF: tetrahydrofolate, PPi: pyrophosphate; Pi: inorganic phosphate.

(A) TYLCCNB imparts characteristic symptoms when co-inoculated with BCTV. N. benthamiana plants were mock inoculated, or inoculated with BCTV or BCTV + TYLCCNB and photographed 21 to 30 days later. (B) DNA gel blot hybridization analysis of BCTV DNA from primary tissue, and BCTV L2-1 and L2-2 DNA from recovered or non-recovered secondary tissue. BCTV and BCTV L2^- mutants were inoculated alone or with TYLCCNB, as indicated. DNA extracts were prepared from pools of at least six plants and digested with ScaI to linearize the viral genome and cleave host genomic 18S rDNA to provide a loading control. Viral single- and double-stranded DNAs (ssDNA, dsDNA) are indicated. (C) PCR detection of BCTV and TYLCCNB DNA in infected plants. An ethidium bromide stained gel of PCR products obtained with TYLCCNB- or BCTV intergenic region (IR)-specific primers is shown. Plants were inoculated as indicated. (D) TYLCCNB complements BCTV L2^- mutants and prevents host recovery. Plants were inoculated with BCTV L2-1 or L2-2 mutants alone, or with TYLCCNB. Symptomatic, primary infected tissues were removed 21-30 days post-inoculation, after which secondary shoots were allowed to grow for 14-21 days. Plants were photographed from above to reveal the extent of recovery in virus inoculated plants, and absence of recovery in co-inoculated plants. (E) Cytosine methylation profiles confirm that TYLCCNB inhibits methylation. Plants were inoculated, and viral DNA was obtained from symptomatic primary tissue, and recovered or non-recovered secondary tissue, as indicated. Methylation of the BCTV IR was assessed by bisulfite sequencing. The circles represent cytosines in the IR and are color coded according to sequence context (CG red, CHH green, CNG blue). Filled circles indicate methylation, and each line represents the sequence of an individual clone. Graphic representations of the same data, with percent methylated cytosines on the Y-axis, are shown to the right. Student's t test was performed on total methylation values using individual clones as data points. Asterisks indicate significant differences between samples with and without TYLCCNB at > 95% confidence.

In experiments with TYLCCNV and PVX, plants were photographed under UV light 14 days post-inoculation. In experiments with BCTV, plants were photographed after 21 days due to the longer latent period of this virus. Results shown are representative of at least three independent experiments with 4 to 8 plants per treatment. (A) N. benthamiana plants containing a transcriptionally silenced GFP transgene (line 16-TGS) were mock inoculated, inoculated with TYLCCNV, or co-inoculated with TYLCCNV and TYLCCNB. (B) Line 16-TGS plants were inoculated with BCTV, BCTV L2-2 (L2^-), or co-inoculated with these viruses and TYLCCNB. (C) Plants were inoculated with the PVX vector, PVX expressing AL2 from CaLCuV (PVX-AL2, positive control), or PVX expressing βC1 (PVX-βC1). GFP expression in the PVX-AL2 (CaLCuV) plant shown was less intense than is typically observed [22]. (D) Gel blot analysis 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 ethidium bromide staining.

(A) Yeast two-hybrid interaction. Bait proteins were expressed as GAL4 DNA binding domain fusions, and prey proteins as GAL4 activation domain fusions, in yeast PJ649A cells. Growth on media lacking leucine (-Leu) and tryptophan (-Trp) indicates maintenance of the bait and prey plasmids. Interaction is indicated by growth on plates also lacking adenine (-Ade), or histidine and adenine (-His-Ade). (B) BiFC interaction. Constructs expressing βC1 and SAHH fused to the N- or C-terminal portions of YFP were delivered to N. benthamiana leaf cells by agroinfiltration. Cells were photographed 48 hours post-infiltration at 40 X magnification using a confocal laser scanning microscope. RFP-histone 2B (RFP-H2B) was used as a marker for the nucleus. The co-expressed proteins are indicated above the photographs, which are representative of results with all possible combinations of fusion proteins. In both yeast two-hybrid and BiFC experiments, DRB4 was co-expressed with DCL4 as a positive control, and βC1 was expressed with DCL4 as a negative control.

N. benthamiana plants containing a transcriptionally silenced GFP transgene (line 16-TGS) were inoculated as indicated and photographed under UV light 14 days post-inoculation. (A) Line 16-TGS plants were inoculated with TYLCCNV, or co-inoculated with TYLCCNV + TYLCCNB (expressing wild-type βC1) or TYLCCNB-3A, expressing βC1 in which lysine residues 49-51 are replaced by alanine (βC1^3A). (B) Line 16-TGS plants were inoculated with the PVX vector, PVX expressing wild-type βC1 (PVX-βC1), or mutant βC1 (PVX-βC1^3A). (C) BiFC interaction. Constructs expressing βC1, βC1^3A, and SAHH fused to the N- or C-terminal portions of YFP were delivered to N. benthamiana leaf cells by agroinfiltration. Cells were photographed 48 hours post-infiltration using a confocal laser scanning microscope. The co-expressed proteins are indicated to the left of the photographs.