Floral stage optimization and immune evasion enhance Agrobacterium-mediated genome editing in Arabidopsis

Agrobacterium-mediated transformation by 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 used a dual-reporter system (RUBY and hygromycin resistance) to identify key floral stages and engineered Agrobacterium strains to evade plant immunity, leading to enhanced transient expression and genome editing (GE). We determined that flowers opened at 6 d 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 showed reduced ovule abortion. Notably, efr mutants exhibited more RUBY-positive ovules and significantly enhanced GE efficiency. Two engineered stealth Agrobacterium strains (AS201 and AS202) expressing a chimeric EF-Tu for evading recognition by EFR enhanced both transient transformation and GE efficiency. 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. By integrating floral stage selection, immune evasion, and Agrobacterium engineering, this study provides a practical and versatile platform to advance plant genome engineering.