The defensin gene NaDLP1, which confers resistance against Alternaria alternata, is transcriptionally regulated by NaWRKY3/6 in Nicotiana attenuata

Plant defensins are ubiquitous across plant species and serve as critical components of the innate immune system against fungal pathogens. In our previous study, we identified NaDLP1, a defensin-like protein 1-encoding gene in wild tobacco (Nicotiana attenuata), which was strongly induced by Alternaria alternata (tobacco pathotype). Virus-induced gene silencing (VIGS) of NaDLP1 compromised resistance, highlighting its essential role in pathogen defense. However, the disease resistance mediated by NaDLP1 and its transcriptional regulation required more detailed studies. Here, we demonstrated that NaDLP1 encoded a typical defensin-like protein and could be secreted into the apoplast space. The recombinant NaDLP1-His protein exhibited potent antifungal activity, inhibiting A. alternata and Botrytis cinerea growth in vitro. Consistent with these findings, NaDLP1-silenced N. attenuata plants (generated via RNA interference (RNAi)) presented increased susceptibility to A. alternata. RNA sequencing (RNA-seq) and Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR) analyses revealed a significant reduction of NaDLP1 transcript levels in NaWRKY3- and NaWRKY6-silenced plants. Electrophoretic mobility shift assay (EMSA) confirmed that both NaWRKY3 and NaWRKY6 directly bound to the W-box motif within the NaDLP1 promoter. Moreover, transient coexpression assays in Nicotiana benthamiana using firefly luciferase (LUC) as a reporter demonstrated that NaWRKY3 and NaWRKY6 functioned as positive regulators of NaDLP1 expression. Collectively, our findings establish NaDLP1 as a key defensin gene conferring resistance against tobacco brown spot disease, with its transcriptional activation mediated by NaWRKY3 and NaWRKY6.