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

O_LIAgrobacterium-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. C_LIO_LIHere, 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. C_LIO_LIWe determined that flowers opened at 6 day-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. Two engineered stealth Agrobacterium strains (AS201 and AS202) expressing a chimeric EF- Tu for evading recognition by EFR enhanced both transient transformation and genome editing 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. C_LIO_LIBy integrating floral stage selection, immune evasion, and Agrobacterium engineering, this study provides a practical and versatile platform to advance plant genome engineering. C_LI