CRISPR/Cas9-targeted mutagenesis of tobacco DMR6 homeologs reduces seedling susceptibility to Phytophthora nicotianae race 0

Phytophthora nicotianae remains a major threat in tobacco production, and resistance based only on dominant resistance genes can be weakened by race specificity. Editing susceptibility genes therefore offers a complementary way to reduce host susceptibility. Here, we identified two DMR6 homeologs in allotetraploid tobacco ( Nicotiana tabacum ), NtDMR6T and NtDMR6S , both of which were induced after P. nicotianae infection. CRISPR/Cas9-mediated mutagenesis in the cultivar Honghuadajinyuan showed that disruption of either gene reduced early seedling susceptibility to P. nicotianae race 0, whereas the double mutant had the lowest disease index under the inoculation conditions tested. The mutants showed no obvious differences in measured plant-size traits in one greenhouse assay and one single-location field trial, although cured-leaf yield and quality were not quantitatively assessed. Transcriptome and RT-qPCR analyses indicated that NtDMR6 loss of function was accompanied by changes in defense-related genes, including genes associated with SA responses, MAPK signaling, peroxidase activity, phenylpropanoid biosynthesis, and sulfur-related metabolism. These responses are consistent with the known role of DMR6 in SA catabolism and are best interpreted as downstream defense-associated changes rather than direct evidence of pathway activation. Overall, these results identify NtDMR6T and NtDMR6S as susceptibility-associated genes in tobacco and provide edited alleles and transcriptomic information that may support future breeding of black shank resistance, although their agronomic and quality performance requires further validation across genetic backgrounds and environments.