A Synthetic Mirtron Platform Enables Stable and Robust Splicing-Dependent Gene Silencing in Plants

Abstract Post-transcriptional gene silencing (PTGS) is widely used for gene function studies and crop improvement; however, conventional transgene-based RNAi and artificial microRNA (amiRNA) approaches are often subject to transgene self-silencing, epigenetic inactivation, viral suppressors of RNA silencing, and regulatory complexity. Here, we establish for the first time a synthetic mirtron platform in plants that defines a splice-gated, non-canonical PTGS architecture, mechanistically distinct from existing RNAi strategies. We demonstrate that precise intron splicing and lariat debranching are essential for target gene silencing, directly coupling pre-mRNA splicing to small RNA-mediated regulation. An optimized mirtron mediates efficient, stable, and heritable silencing of PHYTOENE DESATURASE in Arabidopsis thaliana and enables multiplex silencing of endogenous AUXIN RESPONSE FACTORS in potato, demonstrating applicability in a major crop species. Moreover, mirtron-mediated gene silencing remains highly effective in the presence of the viral suppressor P19, unlike canonical amiRNA-based silencing, highlighting its greater resistance to viral suppression and utility for host-induced gene silencing. Because mirtrons are embedded within endogenous introns, they can be co-expressed with host genes, inheriting their native spatial and temporal expression patterns. When precisely introduced, this architecture supports regulatory outcomes similar to those of gene-edited products that lack foreign DNA. Together, these findings define mirtrons as a compact, stable, and application-ready PTGS platform useful for studying gene regulation and crop biotechnology.