Isolation of an ARGONAUTE gene in pelargonium and identification of candidate genes regulated through ARGONAUTE4-dependent RNA-dependent DNA methylation in Arabidopsis

RNAi-induced gene silencing plays a role in plant DNA methylation and defense. While most gene silencing studies have been performed on annuals, little is known about the expression of key components of this process (like ARGONAUTE proteins) in ornamentals. Using a combination of polymerase chain reaction techniques, an ARGONAUTE4 gene, PhAGO4, was isolated from Pelargonium. PhAGO4 encodes a predicted product of 934 amino acids that contains the PAZ and PIWI domains typical of ARGONAUTE (AGO) proteins. Phylogenetic analyses indicate that PhAGO4 clusters with other plant AGO4 proteins. Organ expression patterns of the AGO4 genes in Pelargonium and Arabidopsis show intriguing differences. AGO4 RNA levels decline with leaf age in both Arabidopsis and Pelargonium . In contrast AGO4 RNA levels in roots relative to leaves are higher in Pelargonium than in Arabidopsis . Both Arabidopsis and Pelargonium AGO4 showed higher RNA levels in flowers than leaves or roots. Even though flowers show higher levels of PhAGO4 RNA when compared to leaves and roots, protein gel blot analysis shows that at the protein level, the reverse is true.RNA interference (RNAi) is a regulatory mechanism found in all eukaryotes that occurs at either the transcriptional or post-transcriptional level. Two key players in Arabidopsis transcriptional gene silencing are small RNAs (sRNAs) and ARGONAUTE (AGO) proteins, specifically AGO4. Therefore, a combination of microarray-based genome-wide transcript profiling, a potential sRNA binding profile, and methylome datasets was used to identify candidate AGO4-regulated protein coding target genes. The highest number of AGO4-associated small RNAs with the potential for binding protein-coding genes showed a preference for either the upstream 1000 bp portion (promoter) or intronic regions. Microarray studies identified 243 up-regulated genes in ago4-1 mutant plants when compared to wild type. Interestingly, 33 up-regulated genes correlated with loci containing potential AGO4-associated sRNA binding sites. AGO4 is thought to recruit enzymes catalyzing non-CG methylation. The majority of potential sRNA binding sites did show a correlation with the non-CG methylation pattern. Taken together, these 33 protein-coding genes are good candidates for direct AGO4 regulation.