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.Methylation of DNA is commonly investigated using methylation-sensitive restriction enzymes and bisulfite sequencing, which converts unmethylated cytosines into uracil. The products are usually amplified by PCR and sequenced after bacterial cloning (14, 23). The critical steps in various protocols are the complete conversion of unmethylated cytosines to uracil (26, 78) and the design of appropriate primers for the converted DNA (reviewed in references 58 and 85). Several bioinformatic tools have been developed to analyze the results (9, 46-48, 66, 71), including recent improvements for the analysis of plant DNA cytosine methylation (27,35).Eukaryotes methylate cytosine at C 5 by methyltransferases, which differ between plants, fungi, and mammals (reviewed in reference 25). In mammals, symmetric CpG sites are usually preferred, whereas nearly every cytosine residue in plant DNA can be methylated (reviewed in reference 5). As a result, only 2 to 8% of mammalian DNA is methylated, compared to up to 50% of DNA in higher plants (reviewed in reference 84). The methylation status is highly dynamic. In plants, demethylation is mediated by DNA glycosylases and strand cleavage (84), a mechanism which would inactivate single-stranded DNA (ssDNA). In addition to C-methylation, N 6 -adenine methylation is well known for bacteria but rarely described for plants (19,74).The circular ssDNA-containing geminiviruses (reviewed in reference 37) are important plant pathogens causing severe damag...

Early Function of the Abutilon Mosaic Virus AC2 Gene as a Replication Brake

“…2b, asterisks) toward the left (more condensed DNA) was absent if HA:AC2 was coexpressed. Such a shift probably represents the addition of a 13th nucleosome to the open minichromosome with 12 nucleosomes, as was shown previously for AbMV DNA in older leaves of Abutilon plants (41,42). However, this particular left shift at later stages of infection was not reproduced in the two subsequently repeated experiments and may be a stochastic rather than a systematic effect under these conditions (46).…”

“…Since nucleosome loading is reflected by the number of superhelical turns per covalently closed circular DNA (cccDNA) molecule (42), the coverage (condensation degree) of minichromosomes can be monitored by determining the topoisomer distribution of cccDNA (46). Moreover, the use of methylation-dependent restriction enzymes (e.g., McrBC) allows the detection of methylation on these specific DNA forms, a prerequisite for the evaluation of geminiviral DNA in this context (41). A regular shift from topoisomers indicating a loading with 12 to 13 nucleosomes was observed previously for AbMVinfected aging leaves of ornamental Abutilon plants under steadystate infection conditions (41,42).…”

“…Geminiviruses build minichromosomes as intermediates for transcription and replication (41)(42)(43)(44), which may be targets of DNA and histone methylation (3,45). Since AC2 has been shown to influence the cellular methyl cycle, it was interesting to investigate whether its overexpression can change the minichromosome architecture by DNA methylation.…”

“…Moreover, the use of methylation-dependent restriction enzymes (e.g., McrBC) allows the detection of methylation on these specific DNA forms, a prerequisite for the evaluation of geminiviral DNA in this context (41). A regular shift from topoisomers indicating a loading with 12 to 13 nucleosomes was observed previously for AbMVinfected aging leaves of ornamental Abutilon plants under steadystate infection conditions (41,42). Since 13 nucleosomes cover the geminiviral 2,632-nucleotide (nt)-long cccDNA completely, whereas 12 nucleosomes leave a gap for the assembly of transcription factors (42), the topoisomer distribution may indicate transcriptionally inactive and active viral genomes, respectively.…”

“…Total nucleic acids from plant tissues were prepared, using extraction buffer (100 mM Tris-HCl [pH 7.0], 100 mM NaCl, 10 mM EDTA, 1% sodium dodecyl sulfate [SDS], 10 mM N-ethylmaleimide [NEM], 100 mM dithiothreitol [DTT]), and phenol-chloroform (twice; 1:1) and chloroform extraction and ethanol precipitation (once) (41). Three hundred nanograms of total DNA per sample was separated by electrophoresis on 1.4% agarose gels supplemented with 10 g/ml chloroquine in the gel as well as in the Trisboric acid-EDTA (TBE) buffer tank and transferred onto Hybond Nϩ membranes (GE Healthcare).…”