Cell-type-specific ablation of siRNAs by Arabidopsis RTL1 reveals a role of phloem companion cells in systemic post-transcriptional gene silencing

Small RNAs regulate numerous biological processes, including intercellular communication and inter-kingdom relationships, yet their tissue-specific functions remain poorly understood. Dissecting these roles requires tools that selectively eliminate small RNAs in defined cell types. Virus-encoded proteins such as P19 have been widely used to neutralize small RNAs owing to their sequestration capacities. However, their restricted binding spectrum to 21-22-nt small RNAs and lack of discrimination between miRNAs and siRNAs limit their utility. Here we show that constitutive expression of RNASE THREE-LIKE1 (RTL1) in Arabidopsis thaliana, previously shown to degrade siRNA precursors but not miRNA precursors, also degrades siRNA duplexes, irrespective of their size, sequence, or sub-cellular localization. Grafting experiments combined with small RNA sequencing revealed that shoot-derived siRNAs accumulate in dcl2 dcl3 dcl4 mutant roots, but are eliminated upon ectopic RTL1 expression, demonstrating that RTL1 efficiently degrades mobile siRNAs in vivo. Because the Arabidopsis endogenous RTL1 is epigenetically silenced in wild-type plants, its ectopic expression under cell-type-specific promoters enables targeted depletion of siRNAs in selected tissues. As a proof of concept, we show that expression of RTL1 in phloem companion cells delays systemic post-transcriptional transgene silencing (PTGS), indicating that mobile siRNAs transit through these cells. Together, these results establish RTL1 as a powerful tool to dissect siRNA mobility pathways and tissue-specific functions.