Adenine methylation at GATC sequences regulates activity of tobacco PR-1 and PR-2 promoters in electroporated protoplasts

Three different constructs, containing promoters of genes which belong to the Pathogenesis Related family (PR-1a, PR-2d) and CaMV 35S promoter, fused with uidA gene (GUS) as a reporter, were tested individually for expression in tobacco protoplasts. DNA containing constructs were amplified in Escherichia coli K12 strains: DH5α (wild type) or GM33 and GM48 defective in methylation systems (Dam and Dam/Dcm mutants, respectively). In contrast to 35S promoter, marked differences in transient activity of both PR promoters were observed depending on the methylation state of the templates. High levels of GUS expression driven by PR-1a or PR-2d were observed only when the template was obtained from the DH5α strain. In vitro DNA methylation with Dam methylase, prior to protoplast transfection, restored high levels of activity to the investigated promoters. A gel mobility shift assay was used to check if there are any differences in binding of PR-1a promoter fragments, containing GATC or GmATC sequences, with tobacco nuclear extract. Our data suggest that N6methyl-deoxyadenine (6-mA), present in GATC sequences, may modulate the regulation of PR-s genes expression.