Integrates JA and ethylene signals in defense against brown spot disease: the role of NaWRKY70

Production of phytoalexins scopoletin and scopolin is regulated by jasmonate (JA) and ethylene signals in Nicotiana species in response to Alternaria alternata, the necrotrophic fungal pathogen causing brown spot disease. However, how these two signals are coordinated to control this process remains unclear. Here, we found that levels of these two phytoalexins and transcripts of their key enzyme gene feruloyl-CoA 6-hydroxylase 1 (NaF6H1) were synergistically induced in wild tobacco N. attenuata by co-treatments of methyl jasmonate (MeJA) and ethephon, but were not altered by treatments with either MeJA or ethephon along. Through co-expression and functional analysis we identified NaWRKY70 as the key regulator for this synergistic induction. NaWRKY70 not only directly bound to NaF6H1 promoter and activated its expression but also served as a regulation node to integrate JA and ethylene signals. Acting like locks, NaJAZe1, NaJAZe2 and NaEBF2 interacted with NaWRKY70 separately to suppress NaF6H1 expression. Meanwhile, NaMYC2b and NaEIN3-like 1functioned as transcription regulators of NaWRKY70 and activated of NaF6H1 expression by protein interaction with NaWRKY70. Finally, NaWRKY70 controlled JA-Ile production by binding and activating its biosynthetic genes. Thus, our data uncovers a novel but complicate regulation network of phytoalexins by two phytohormonal signals, and demonstrates that NaWRKY70 integrates both JA and ethylene signals through transcriptional regulation and protein interaction to regulate scopoletin and scopolin biosynthesis and plant resistance to A. alternata.