RLL5, an F-box-containing protein, involved in preventing transgene silencing and in maintaining global DNA methylation in Arabidopsis.

RNA-directed DNA methylation (RdDM) and ROS1-dependent active DNA demethylation pathways are antagonistic processes that dynamically regulate site-specific methylation. In this study, we obtained a mutant with reduced luciferase (LUC) luminescence by genetic screening, which was named rll5-1 (for reduced LUC luminescence 5-1). The rll5-1 mutant showed narrower, frizzled and curly leaves, and the low-LUC-luminescence phenotype in the rll5-1 mutant can be largely restored by DNA methylation inhibitor 5-Aza-2'-deoxycytidine. Map-based cloning coupled with genome resequencing data revealed that a nucleotide substitution of G to A was found at the 124th bp of ORF of At4G10190, leading to an aspartate-to-asparagine change at position 42 in such a protein. Bisulfite sequencing data indicated that DNA methylation of 3' region of the double 35S promoter that drives the LUC expression was appreciably increased. Further analysis revealed that there were 4747 hypo-DMRs and 936 hyper-DMRs found in the rll5-1 genome, and the hypo-DMRs was predominantly distributed on TEs, which appeared to stem from the downregulation of a few RdDM pathway genes and DNA methyltransferase genes. Closer inspection demonstrated that there were 1229 hypo-DMRs commonly shared among rll5-1, nrpd1-3 and nrpe1-11, and a total of 1349 hypo-DMRs were common to rll5-1 and cmt2 mutants. Thus, these studies demonstrate the roles of RLL5 in preventing transgene silencing and in maintaining genome-wide DNA methylation in a direct/indirect or locus-specific manner.