NaWRKY3 is a master transcriptional regulator of defense network against Alternaria alternata in Nicotiana attenuata

WRKY transcription factors are involved in plant defense responses against pathogens. However, no WRKYs have been reported yet in resistance of Nicotiana species to Alternaria alternata, a necrotrophic fungal pathogen causing brown spot disease. Here, we found that silencing NaWRKY3 lead to wild tobacco Nicotiana attenuata highly susceptible to A. alternata. Combination of transcriptome, electrophoretic mobility shift, ChIP-qPCR and dual-LUC analyses, we uncovered that NaWRKY3 bound to many defense genes promoter and activated their expression. Target genes included: 1) lipoxygenases 3, ACC synthase 1 and ACC oxidase 1, three key enzyme genes for JA and ethylene biosynthesis which were critical for A. alternata resistance; 2) feruloyl-CoA 6-hydroxylase 1 (NaF6H1), the key enzyme gene for phytoalexins against A. alternata, scopoletin and scopolin; and 3) three A. alternata resistance genes, long non-coding RNA (LncRNA L2), NADPH oxidase (NaRboh D) and berberine bridge-like (NaBBL28). Silencing LncRNA L2 reduced A. alternata-induced levels of JA and NaF6H1 expression. NaRboh D-silenced plants were strongly impaired in ROS production and stomata closure responses. NaBBL28 was the first A. alternata resistance BBLs identified and was involved in HGL-DTGs hydroxylation. Finally, NaWRKY3 could bind to its own promoter but acted as a transcriptional repressor. Thus we demonstrated that NaWRKY3 is a fine-tuned master regulator of defense network against A. alternata in N. attenuata by regulating different signaling pathways and defense metabolites. For the first time, such an important WRKY was identified in Nicotiana species, providing new insight into defense mechanism of Nicotiana plants to A. alternata.