Effect of CpG methylation on gene expression in transfected plant protoplasts

Activity of the cat gene driven by the cauliflower mosaic virus 35S promoter has been assayed by transfecting petunia protoplasts with the pUC8CaMVCAT plasmid. In vitro methylation of this plasmid with M · HpaII (methylates Ċ CĊGG sites) and M · HhaI (methylates GĊGC sites) did not affect bacterial chloramphenicol acetyltransferase (CAT) activity. It should be noted, however, that no HpaII or HhaI sites are present in the promoter sequence. In contrast, in vitro methylation of the plasmid with the spiroplasma methylase M · SssI, which methylates all ĊpG sites, resulted in complete inhibition of CAT activity. The promoter sequence contains 16 CpG sites and 13 CpNpG sites that are known to be methylation sites in plant DNA. In the light of this fact, and considering the results of the experiments presented here, we conclude that methylation at all ĊpG sites leaving CpNpG sites unmethylated is sufficient to block gene activity in a plant cell. Methylation of ĊpNpG sites in plant cells may, therefore, play a role other than gene silencing.