DNA demethylases function in conjunction with DNA methyltransferases to modulate genomic DNA methylation levels in plants. The Arabidopsis genome contains four DNA demethylase genes, DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1) also known as DEMETER-LIKE 1 (DML1), DML2, and DML3. While ROS1, DML2, and DML3 were shown to function in disease response in somatic tissues, DME has been thought to function only in reproductive tissues to maintain the maternal-specific expression pattern of a subset of imprinted genes. Here we used promoter:β-glucuronidase (GUS) fusion constructs to show that DME is constitutively expressed throughout the plant, and that ROS1, DML2, and DML3 have tissue-specific expression patterns. Loss-of-function mutations in DME cause seed abortion and therefore viable DME mutants are not available for gene function analysis. We knocked down DME expression in a triple ros1 dml2 dml3 (rdd) mutant background using green tissue-specific expression of a hairpin RNA transgene (RNAi), generating a viable ‘quadruple’ demethylase mutant line. We show that this rdd DME RNAi line has enhanced disease susceptibility to Fusarium oxysporum infection compared to the rdd triple mutant. Furthermore, several defence-related genes, previously shown to be repressed in rdd, were further repressed in the rdd DME RNAi plants. DNA methylation analysis of two of these genes revealed increased differential promoter DNA methylation in rdd DME RNAi plants compared to WT, beyond the difference observed in the parental rdd plants. These results indicate that DME contributes to DNA demethylase activity and disease response in somatic tissues.
