Functional allelic diversity of PTGS components revealed by a high-throughput RUBY-based screening.

Post-transcriptional gene silencing (PTGS) mediated by small RNA significantly impairs both transgene efficiency and endogenous gene expression. Although multiple PTGS components have been identified through forward genetic screening using various reporter systems, mutations reported for these known PTGS components remain limited, hindering our understanding of the underlying mechanisms and the functional roles of specific protein domains. Here, we combined the 5'-3' RNA decay mutant ein5-1 with a non-invasive RUBY system to perform high-throughput screening for mutants that can restore RUBY expression. We identified 239 mutations covering almost all critical protein domains in key PTGS components and promising novel factors. Through systematic phenotypic evaluation, we found spatial-temporal repression of PTGS in distinct mutants. Mutations affecting enzymatic/catalytic activity center, RNA-binding sites, or interactions between essential PTGS components dramatically reduced RUBY-derived siRNA production. These mutant alleles provide valuable genetic resources for investigating domain-specific functions of known PTGS factors, thereby offering support for fine-tuning PTGS efficiency.