Plants can retard virus infection by means of TGS and PTGS, but these defense mechanisms may be counter-balanced by viral suppressors of silencing and replicative evasion of DNA methylation (Csorba et al., 2009; Pooggin, 2013; Raja et al., 2010; Rodriguez-Negrete et al., 2009; Voinnet, 2005). Plants employ symmetric DNA methylation at CG or CNG sites like other eukaryotic cells, but produce higher levels of asymmetric DNA methylation at CHH sites (H: no guanine). Different methyltransferases are involved in these DNA modifications: de novo methylation at CHH sites is caused by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2, or its homolog DRM1), whereas maintenance methylation is achieved by CHROMOMETHYLASE 3 (CMT3; maintains the methylation at CHG), METHYLTRANSFERASE 1 (MET1; for the maintenance of CG methylation) and KRYPTONITE (KYP2; for DNA methylation maintenance at CHH and H3K9 methylation) (Raja et al., 2010).<br />For geminiviruses, <a href='http://www.mk-2206.com/viewtopic.php?f=2&t=7199
'>PI3K Akt mTOR Compound Library</a> methylation of the viral minichromosomes is one possible host defense mechanism, which is counter-balanced by different suppressor proteins, depending on the virus genus or species. Certain geminiviral gene products (AL2 or L2; syn. AC2, C2) influence, amongst other functions, the methyl cycle of the host (Raja et al., 2010). These proteins target enzymes in the S-adenosylmethionine (SAM) pathway (Buchmann et al., 2009; Zhang et al., 2011). Similarly, the ßC1 protein of a satellite DNA which is associated with the tomato yellow leaf curl China virus (TYLCCNV) was found to interact with S-adenosyl homocysteine hydrolase (SAHH) (Yang et al., 2011). Additionally, a different strategy to suppress silencing was discovered for geminiviral Rep and C4 (Rodriguez-Negrete et al., 2013) which was able to downregulate MET1 and CMT3 and prevent maintenance of de novo methylation at CG and CHG sites.<br />It has been proposed that geminiviruses can evade repressive cytosine methylation and transcriptiona<img src="http://www.PRECISIONFDA.ORG/image/1-s2.0-S1871403X15002033-gr3.jpg#
" />l silencing by the aid of RCR and RDR (Paprotka et al., 2011; Pooggin, 2013). Previous results have shown that heterogeneous linear (lin) and multimeric (mult) dsDNAs were the main targets of methylation, whereas circular monomeric dsDNA was hardly affected (Paprotka et al., 2011). Bisulfite sequencing-based analyses were compatible with a stochastic distribution of methylated sites in the viral DNAs, although their quantification power was regarded critically in the previous report (Paprotka et al., 2011). Recently determined high levels of C>T mutations in the viral gene pool, which is the same diagnostic exchange in the bisulfite sequencing, add a further argument towards a cautious interpretation of this data (Richter et al., 2016a, 2016b). In spite of these limitations, the available results hint at a role of methylation in host defense to suppress geminiviral gene expression, most prominent during recovery from infection (Buchmann et al., 2009; Ceniceros-Ojeda et al., 2016; Chung and Sunter, 2014; Chung et al., 2014; Liu et al., 2014; Raja et al., 2014, 2008; Rodriguez-Negrete et al., 2013, 2009; Yang et al., 2011).<br />In order to circumvent the limitations of bisulfite-sequencing and the use of restriction enzymes that are impaired by DNA methylation, we used DNA methylation-dependent restriction enzymes (MdRs) as a complementary diagnostic tool (Paprotka et al., 2011). MdRs can cleave a DNA only if a strand is methylated and introduce a double strand cut (Horton et al., 2012; Loenen and Raleigh, 2014). Intriguingly, one major product of MdR <a href='http://en.wikipedia.org/wiki/Digestion
'>digestion</a> of geminiviral <img src="http://farm5.static.flickr.com/4432/37139001181_1bde3d3d56.jpg
" align="left" width="281" style="padding:10px;"/> DNA was a lin dsDNA of genomic length indicating that either circular monomeric cccDNA was cut stochastically only once, or that the heterogeneous lin dsDNA - a prominent replication intermediate consisting of concatemeric viral genome units - was cut at a precise position (Paprotka et al., 2011). We can now provide the first evidence that a specific DNA form of monomeric cccDNA with 12 superhelical turns (indicative of minichromosomes with 12 nucleosomes) was preferentially methylated. Furthermore, two specific sites, interestingly flanking the CR sequence, were identified by conventional cloning and sequencing. Circomics (Wyant et al., 2012), however, using a newly developed method of ligation-mediated rolling circle amplification (RCA) combined with Illumina sequencing, revealed many more MdR sites with a preference for an inter-site distance of nucleosomal DNA length scattered throughout the whole genome.