A method for maintaining the release of co-suppression and maximally restoring the RDR6 expression.

Key message: Tissue-specific RDR6 compensation rescues Arabidopsis defects while maintaining seed polyunsaturated fatty acid accumulation, balancing co-suppression relief and key trait retention for modular engineering. The transgene-induced co-suppression of fatty acid desaturase 2 (FAD2) can be effectively released in rdr6 mutant, enabling a significant increase in polyunsaturated fatty acid (PUFA) content in seeds. However, the global suppression of RNA-dependent RNA polymerase 6 (RDR6) compromises plant growth and disease resistance. To address this limitation, we developed a spatiotemporal compensation strategy by restoring RDR6 expression in non-seed tissues using tissue-specific promoters while maintaining its low expression during seed maturation. To implement this goal, we identified P_BnTC06, a Brassica napus promoter, through transcriptomic data mining and functional characterization. GUS staining revealed that the P_BnTC06 promoter drives strong gene expression in vegetative tissues (e.g., leaves, stems, and flowers) but exhibits negligible activity in mid- to late-stage-developing seeds. We introduced P_BnTC06::RDR6 into Pha::AtFAD2/rdr6-11, the previously established high- PUFA Arabidopsis line. This intervention rescued the rdr6 mutant phenotype (characterized by gracile, downward-curling leaves) to wild-type morphology and restored RDR6 expression across non-seed tissues, while maintaining minimal expression in middle and late developing seeds. Crucially, FAD2 transcript levels remained at a high level during late seed development, resulting in sustained high PUFA accumulation in mature seeds. This strategy establishes a practical strategy to circumvent transgene co-suppression and proposes a modular framework for precision breeding of complex traits in crops.