Abstract
DNA methylation maintains genome stability and regulates gene expression in plants. RNA-directed DNA methylation (RdDM) is critical for appropriate methylation. However, no efficient tools are available for the investigation of the functions of specific DNA methylation. In this study, the cucumber mosaic virus vector was used for targeted DNA methylation. Methylation was rapidly induced but gradually decreased from the 3' end of the target endogenous sequence in Nicotiana benthamiana, suggesting a mechanism to protect against the ectopic introduction of DNA methylation. Increasing 24-nt siRNAs blocked this reduction in methylation by down-regulating DCL2 and DCL4. RdDM relies on the sequence identity between RNA and genomic DNA; however, this identity does not appear to be the sole determinant for efficient DNA methylation. The current findings provide new insight into the regulation of DNA methylation and promote additional effort to develop efficient targeted DNA methylation in plants.