CRISPR/Cas9-Targeted Mutagenesis of Arabidopsis <em>DMR6 </em>Orthologs in Tobacco Confers Resistance to <em>Phytophthora nicotianae</em>

Phytophthora nicotianae is an oomycete pathogen that severely threatens tobacco production worldwide. Though several dominant resistance (R) genes against P. nicotianae are used in tobacco breeding, they often fail due to rapid emergence of new virulent strains. Instead, targeting plant susceptibility (S) genes offers a promising approach for durable and broad resistance. Evidence from various plant species demonstrates that loss of the S gene DMR6 enhances disease resistance without compromising yield, emphasizing its value for resistance breeding. In this study, we identified and functionally characterized two DMR6 orthologs in tobacco (Nicotianan tobaccum), NtDMR6T and NtDMR6S, which were both induced upon P. nicotianae infection. Targeted mutagenesis of NtDMR6T and NtDMR6S using CRISPR/Cas9 demonstrated that single-gene knockouts conferred enhanced resistance to P. nicotianae, while double mutants exhibited an additive resistance effect. Notably, all mutant lines showed no obvious growth or developmental defects under greenhouse or field conditions. RT-qPCR analysis indicated that NtDMR6s negatively regulate tobacco resistance by modulating multiple defense pathways, including the MAPK signaling cascade. Collectively, our findings demonstrate that NtDMR6T and NtDMR6S act as negative regulators of resistance in allotetraploid tobacco and represent promising S gene targets for the development of P. nicotianae resistant cultivars, thereby providing a new strategy for tobacco disease resistance breeding.