Exogenous 24nt siRNAs induce AGO4A-dependent silencing via promoter DNA methylation and H3K9me2 deposition

Transcriptional Gene Silencing (TGS) is an essential process in plants for development, gene regulation, defense against viruses, and genome integrity. TGS is predominantly established by RNA-directed DNA methylation (RdDM), a self-reinforcing mechanism in which sRNAs guide transcriptional suppressors to target genomic loci by sequence complementarity, and methylated DNA in turn facilitates sRNA genesis. Recently, exogenous application of promoter targeting long dsRNAs was associated with promoter methylation without any detectable gene silencing. A plethora of sRNAs of different sizes and types form as cleavage products of precursor dsRNAs such as pre-miRNAs, inverted-repeats, viral replication intermediates, or exogenous dsRNAs. Due to the complexity of sRNA products, the features of the sRNAs, which trigger de novo RdDM, remain enigmatic. Here, we demonstrated that in planta delivery of chemically synthesized 24-nucleotide(nt) small interfering RNAs (siRNAs), targeting the 35S promoter of GFP-expressing Nicotiana benthamiana (Nb) 16c line, was sufficient to induce RdDM, H3K9me2 deposition, and also TGS. Using CRISPR/Cas-mediated gene editing, we showed that exogenous 24nt siRNA-triggered TGS is dependent on ARGONAUTE 4A (NbAGO4A) but not on NbAGO4B. Exogenously administered 24nt siRNAs could provide the means to investigate such initiation events, while allowing functional dissection of siRNA classes and their modifications in planta.