Floral Stage Optimization and Immune Evasion Enhance Agrobacterium -Mediated Genome Editing in Arabidopsis

Agrobacterium -mediated transformation via floral inoculation (AMT-FI) enables genetic engineering without tissue culture. It is widely used in the model plant Arabidopsis thaliana , yet its efficiency and broader applicability remain limited. Here, we identify key floral stages and develop novel strategies to enhance AMT-FI-based genome editing.

Using a dual-reporter system (RUBY and hygromycin resistance), we determined that flowers opened at 6 days post-inoculation (DPI) are optimal for high transformation efficiency, with nearly 100% of siliques harboring transformants. However, Agrobacterium infection induced ovule abortion, particularly in wild-type (Col-0) plants, whereas efr mutants lacking the EF-Tu receptor (EFR)-mediated pattern-triggered immunity (PTI) showed reduced ovule abortion. Notably, efr mutants exhibited more RUBY-positive ovules and significantly enhanced genome editing efficiency, despite showing no improvement in stable transformation rates. These results suggest that PTI restricts transient transformation rather than T-DNA integration. We further engineered stealth Agrobacterium strains expressing a chimeric EF-Tu for evading recognition by EFR. These GV3101-derived strains, named as AS201 and AS202, enhanced both transient transformation and genome editing efficiency without significantly altering stable transformation frequency. Remarkably, genome-edited T1 plants could be recovered based on phenotype or direct sequencing without the need for antibiotic selection when targeting flowers opened at 6 DPI. Our findings reveal EFR-mediated immunity as a key barrier to efficient transient transformation and genome editing via AMT-FI. By integrating floral stage selection, immune evasion, and Agrobacterium engineering, this study provides a practical and versatile platform to advance plant genome engineering.