NaWRKY3 is a master transcriptional regulator of the defense network against brown spot disease in wild tobacco.

WRKY transcription factors are involved in plant defense against pathogens. However, no WRKYs have been reported to be involved in resistance to tobacco brown spot disease caused by Alternaria alternata. Here, we found that NaWRKY3 plays a critical role in Nicotiana attenuata against A. alternata. It bound and regulated many defense genes, including: 1) lipoxygenases 3, ACC synthase 1 and ACC oxidase 1, three JA and ethylene biosynthetic genes for A. alternata resistance; 2) feruloyl-CoA 6'-hydroxylase 1 (NaF6'H1), the biosynthetic gene for the phytoalexins scopoletin and scopolin; and 3) three A. alternata resistance genes, the long non-coding RNA L2, NADPH oxidase (NaRboh D) and berberine bridge-like (NaBBL28). Silencing L2 reduced JA levels and NaF6'H1 expression. NaRboh D-silenced plants were severely impaired in ROS production and stomatal closure responses. NaBBL28 was the first A. alternata resistance BBL identified and was involved in the hydroxylation of HGL-DTGs. Finally, NaWRKY3 bound to its own promoter but repressed its expression. Thus we demonstrated that NaWRKY3 is a fine-tuned master regulator of the defense network against A. alternata in N. attenuata by regulating several signaling pathways and defense metabolites. This is for the first time that such an important WRKY has been identified in Nicotiana species, providing new insights into defense against A. alternata.