Conformation-selective methylation of geminivirus DNA

Abstract

Geminiviruses with small circular single-stranded DNA genomes replicate in plant cell nuclei by using various double-stranded DNA (dsDNA) intermediates: distinct open circular and covalently closed circular as well as heterogeneous linear DNA. Their DNA may be methylated partially at cytosine residues, as detected previously by bisulfite sequencing and subsequent PCR. In order to determine the methylation patterns of the circular molecules, the DNAs of tomato yellow leaf curl Sardinia virus (TYLCSV) and Abutilon mosaic virus were investigated utilizing bisulfite treatment followed by rolling circle amplification. Shotgun sequencing of the products yielded a randomly distributed 50% rate of C maintenance after the bisulfite reaction for both viruses. However, controls with unmethylated single-stranded bacteriophage DNA resulted in the same level of C maintenance. Only one short DNA stretch within the C2/C3 promoter of TYLCSV showed hyperprotection of C, with the protection rate exceeding the threshold of the mean value plus 1 standard deviation. Similarly, the use of methylation-sensitive restriction enzymes suggested that geminiviruses escape silencing by methylation very efficiently, by either a rolling circle or recombination-dependent replication mode. In contrast, attempts to detect methylated bases positively by using methylcytosine-specific antibodies detected methylated DNA only in heterogeneous linear dsDNA, and methylation-dependent restriction enzymes revealed that the viral heterogeneous linear dsDNA was methylated preferentially.

Fig. 1.

Methylation-sensitive restriction enzyme digestion of viral DNA by use of isoschizomers. Total nucleic acids (300 ng DNA [each]) from N. benthamiana plants infected systemically with AbMV or TYLCSV and harvested at 14, 21, or 49 dpi were digested with two sets of isoschizomers: MspI (blocked by cytosine methylation of the external C) with HpaII (blocked by each of the cytosine methylations) (at CCGG) and Sau3AI (blocked by cytosine methylation but not by adenine methylation) with MboI (blocked by cytosine as well as adenine methylation) (at GATC). Untreated samples (U) with the same amounts of DNA were loaded in parallel. The samples were electrophoresed in 1.4% agarose gels (3 h, 120 V), stained with ethidium bromide (a and c), blotted onto nylon membranes, and detected with virus-specific full-length probes for TYLCSV (b) and AbMV A (d). RCA products (1 μl; diluted 1:50) from correspondingly infected plants were digested with the respective enzymes and served as size markers for completely unmethylated DNA fragments (C). Hybridization standards (M) were 1, 10, or 100 pg of linearized full-length dsDNA fragments (lin) of AbMV or TYLCSV. In order to control the completeness of digestion, 300 ng of lambda DNA was supplied to each enzyme reaction mix, forming band patterns (λp) in ethidium bromide-stained gels. Undigested λ DNA was expected to migrate to the same position as genomic plant DNA (λ/g). Geminivirus fragments which were not detected in the controls are marked by black and white asterisks. Expected fragment sizes (in base pairs) of the respective digestion products, calculated from the sequences, are shown to the left and right of the panels. Note that ssDNA tends to smear and to create more diffuse bands than those of dsDNA, as seen after Southern blot hybridization. Bands in undigested samples of ethidium bromide-stained gels refer to the usual genomic DNA and host RNA species (not indicated).

Fig. 2.

RCA/RFLP analysis of bisulfite-treated DNA. The genome map of TYLCSV shows DraI sites which are present in the source DNA (underlined) or which may be created on the viral (v) or complementary (c) strand after bisulfite treatment. Total nucleic acids from systemically TYLCSV-infected N. benthamiana plants at 9, 14, 20, and 35 dpi were converted by bisulfite treatment and amplified by RCA. Three-microliter samples of these products were digested with HpaII or DraI, separated in 1.4% agarose gels (3 h, 120 V), and then stained with ethidium bromide (a) and Southern blotted (b). As controls (C), similarly digested RCA products (1 μl) of untreated samples were applied. The gels were blotted onto nylon membranes and detected with a full-length virus DNA probe specific for TYLCSV. The remaining fragments of unconverted molecules are marked by asterisks. No RCA product was obtained in the sample from 9 dpi.

Fig. 3.

Analysis of AbMV DNA as described in the legend to Fig. 2, but with detection with a full-length virus DNA probe specific for AbMV DNA A.

Fig. 4.

Overall cytosine methylation of the TYLCSV viral strand and AbMV DNA B complementary strand. (a) Distributions of fragments obtained after sequencing of DraI-digested randomly cloned RCA products. Positions of the ORFs are marked with arrows. (b) Frequencies of protected C's in classes of 100 nt (%C) are represented by bars, and the threshold value for unmethylated phage DNA is shown as a dashed line. (c) Distribution of frequencies (f) for all class results. (d) An exceptionally protected stretch of TYLCSV is exemplified for the viral (v) and complementary (c) strands.

Fig. 5.

Methylation-dependent restriction enzyme digestion of AbMV (a)- or TYLCSV (b)-infected N. benthamiana plants (P#, plant numbers) harvested at the indicated dpi. (c) In addition, individual leaves (L#, starting with the youngest leaf as L1) of ornamental Abutilon plants which were naturally infected with AbMV were collected. Total nucleic acids (60 ng DNA [each]) were digested with McrBC (E+) or not digested (E−), separated electrophoretically in 1.4% agarose gels containing chloroquine (50 μg/ml [a] or 20 μg/ml [b and c]), and analyzed by Southern blot hybridization against the respective probes (AbMV DNA A or TYLSCV lacking each intergenic region). Viral DNA forms are indicated as described in the legend to Fig. 1 and also as multimeric (m), open circular (oc), or relaxed covalently closed circular (rccc) DNA. As a loading control and to ensure complete digestion, equal amounts of every sample were separated in parallel in 1.4% agarose gels and stained with ethidium bromide to show the genomic plant DNA. Hybridization standards (M) were 100 pg (a) or 1, 10, or 100 pg (b and c) of linearized full-length dsDNA fragments (lin) of AbMV DNA A or TYLCSV. For each time point, two of five plants were selected randomly and used for digestion (a and b).

Fig. 6.

Positive detection of methylated viral DNA after 2D agarose gel electrophoresis. Following Southern blotting, either AbMV DNA was probed with full-length DNA A (a) or methylated DNA was visualized with m5C antibody (b). In an independent experiment, viral DNA (100 ng total nucleic acid from AbMV-infected N. benthamiana plants harvested at 21 dpi) was kept untreated (c) or treated with McrBC (d) and then analyzed by 2D agarose gel electrophoresis (first dimension, 0.3% SDS; second dimension, 20 μg/ml chloroquine; 19 h at 45 V). Southern blots were hybridized (c and d) with a probe based on AbMV DNA A lacking the intergenic region. The most prominent viral DNA forms are as described in the legend to Fig. 5 and also included heterogeneous linear dsDNA (h and h′), recombination-dependent replication intermediates (RDR), complementary strand replication intermediates (CSR), and dimeric forms (2×).