Nivedita Singh, Shambhavi Dwivedi, Deeksha Singh, Pranshu Kumar Pathak, Shuchi Srivastava, Prabodh Kumar Trivedi
ABSTRACT Anthocyanins and lignin, both derived from the phenylpropanoid pathway, play essential roles in plant defence and development. While anthocyanins attract pollinators and provide antioxidative protection under stress, lignin contributes to structural integrity, vascular function, and pathogen resistance. R2R3‐MYB transcription factors are key regulators of these pathways, functioning as both activators and repressors. Here, we functionally characterised NtMYB308, a tobacco R2R3‐MYB transcription factor containing a bHLH‐interaction motif and an EAR repression domain. Virus‐induced gene silencing (VIGS) and promoter‐binding assays demonstrated that NtMYB308 acts as a transcriptional repressor of anthocyanin and lignin biosynthetic genes. CRISPR/Cas9‐generated knockout lines ( NtMYB308 CR ) exhibited elevated anthocyanin accumulation and increased lignin deposition, whereas overexpression lines ( NtMYB308OX ) showed reduced levels of both metabolites. Notably, NtMYB308 CR plants displayed increased resistance towards fungal pathogens Alternaria solani and Rhizoctonia solani , likely due to reinforced cell walls and elevated antioxidant capacity. In contrast, NtMYB308OX plants were more susceptible. These findings establish NtMYB308 as a key negative regulator of phenylpropanoid metabolism and biotic stress tolerance, offering a potential target for genetic manipulation to enhance disease resistance and reduce reliance on chemical pesticides, thereby promoting sustainable crop production and environmental health.