Role of Arabidopsis AGO6 in siRNA accumulation, DNA methylation and transcriptional gene silencing

Argonautes (AGOs) are conserved proteins that contain an RNA-binding PAZ domain and an RNase H-like PIWI domain. In Arabidopsis, except for AGO1, AGO4 and AGO7, the roles of seven other AGOs in gene silencing are not known. We found that a mutation in AGO6 partially suppresses transcriptional gene silencing in the DNA demethylase mutant ros1-1. In ago6-1ros1-1 plants, RD29A promoter short interfering RNAs (siRNAs) are less abundant, and cytosine methylation at both transgenic and endogenous RD29A promoters is reduced, compared to that in ros1-1. Interestingly, the ago4-1 mutation has a stronger suppression of the transcriptional silencing phenotype of ros1-1 mutant. Analysis of cytosine methylation at the endogenous MEA-ISR, AtREP2 and SIMPLEHAT2 loci revealed that the CpNpG and asymmetric methylation levels are lower in either of the ago6-1 and ago4-1 single mutants than those in the wild type, and the levels are the lowest in the ago6-1ago4-1 double mutant. These results suggest that AGO6 is important for the accumulation of specific heterochromatin-related siRNAs, and for DNA methylation and transcriptional gene silencing, this function is partly redundant with AGO4.