Are ta-siRNAs only originated from the cleavage site of miRNA on its target RNAs and phased in 21-nt increments?

• The 22 nt ta-siR2140 was unfitted for the canonical ta-siRNA biogenesis pathway.
• Thousands of phased siRNAs unfitted for the canonical model were also identified.
• 110 novel phased siRNA–target interactions were validated.
• The miR173-directed gene regulatory network in Arabidopsis was reconstructed.
• A revised model for ta-siRNA biogenesis was proposed.

Trans-acting siRNAs (ta-siRNAs) are a class of small RNAs playing crucial roles in the regulation of plant gene expression. According to the canonical model, specific miRNA-guided cleavage of a TAS transcript triggers and sets the registry for the subsequent production of ta-siRNAs at 21-nt increments from the cleavage site. However, a previously validated 22-nt ta-siR2140 indicated that ta-siRNAs might be initiated from other phase increments and registers, which resulted in massive ta-siRNAs missing in the canonical model. To test this hypothesis, we employed high-throughput sequencing data to thoroughly identify the miR173-triggered ta-siRNAs from TAS1/TAS2 transcripts. As a result, thousands of phased siRNAs not generated through the canonical pathway were identified and 110 novel siRNA—target interactions were further validated based on degradome sequencing data. Based on these results, we propose that the canonical biogenesis model of ta-siRNAs should be modified in order to recruit the previously unidentified ta-siRNA candidates.