Methylation of nonintegrated multiple copy DNA in plants

DNA sequences present in multiple copies in plant genomes are often methylated. However, it was not known if methylation occurs on multiple copy DNA molecules that are not integrated into the plant genome. To investigate this possibility, the methylation state of cauliflower mosaic virus [CaMV] DNA was studied in inoculated turnip leaves at different days post-inoculation [DPI]. Unencapsidated CaMV DNA was found in an unmethylated state up to 7 DPI. By 9 DPI, viral DNA was methylated at almost all HpaII/MspI sites within the CaMV genome. DNA methylation did not appear to be preferential but occurred at almost all HpaII/MspI sites at approximately the same time. Methylation appeared to occur in an all or none manner, suggestive of a switch mechanism. These data strongly suggest that copy number-dependent methylation occurs on DNA molecules that are not integrated into the genome of a host cell.