De novo DNA methylation in Arabidopsis thaliana is catalyzed by the 
methyltransferase DRM2, a homolog of the mammalian de novo methyltransferase 
DNMT3. DRM2 is targeted to DNA by small interfering RNAs (siRNAs) in a process 
known as RNA-directed DNA Methylation (RdDM). While several components of the 
RdDM pathway are known, a functional understanding of the underlying mechanism 
is far from complete. We employed both forward and reverse genetic approaches to 
identify factors involved in de novo methylation. We utilized the FWA transgene, 
which is methylated and silenced when transformed into wild-type plants, but 
unmethylated and expressed when transformed into de novo methylation mutants. 
Expression of FWA is marked by a late flowering phenotype, which is easily 
scored in mutant versus wild-type plants. By reverse genetics we discovered the 
requirement for known RdDM effectors AGO6 and NRPE5a for efficient de novo 
methylation. A forward genetic approach uncovered alleles of several components 
of the RdDM pathway, including alleles of clsy1, ktf1, and nrpd/e2, which have 
not been previously shown to be required for the initial establishment of DNA 
methylation. Mutations were mapped and genes cloned by both traditional and 
whole genome sequencing approaches. The methodologies and the mutant alleles 
discovered will be instrumental in further studies of de novo DNA methylation.