Resistance breeding in elite hybrid rice by EBE-editing for bacterial blight

Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is devastative to hybrid rice. Engineering effector-binding elements (EBEs) in susceptibility gene promoters confers resistance; however, its application in elite restorer and sterile lines remains largely unexplored, limiting the systematic deployment of these traits into commercial hybrid cultivars. In this study, we evaluated seven representative Xoo strains against elite parents Huazhan and Longke638S (LK638S). While Huazhan was fully susceptible, LK638S and their F 1 hybrids (LHZ) exhibited resistance to LN18, indicating the presence of a dominant, strain-specific resistance ( R ) gene. Therefore, we simultaneously edited the EBEs of three OsSWEET genes in both parents using CRISPR-Cas9. The resulting edited lines, H134R and LK638R, were crossed to produce F 1 and F 2 populations. Comprehensive analysis revealed no off-target mutations or altered agronomic performance in the hybrid lines. Notably, in-silico, in-vitro, and in-vivo assays demonstrated that higher base-pair deletions conferred resistance against the tested Xoo strains. Interestingly, thymine insertion alone was found insufficient to block transcription activator-like effector (TALE) mediated susceptibility, as structural flexibility allows TALEs to tolerate a few mismatches. Additionally, the obtained F 2 population followed Mendelian inheritance. Collectively, this work suggests effective EBE mutation and bridges the gap between genome editing and hybrid breeding by demonstrating that multiplex EBE editing in elite parental lines can be translated into stable hybrid rice.