NaWRKY3 is a master transcriptional regulator of defense network againstAlternaria alternatainNicotiana attenuata

Abstract

AbstractWRKY transcription factors are involved in plant defense responses against pathogens. However, no WRKYs have been reported yet in resistance ofNicotianaspecies toAlternaria alternata, a necrotrophic fungal pathogen causing brown spot disease. Here, we found that silencingNaWRKY3lead to wild tobaccoNicotiana attenuatahighly susceptible toA. 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 1andACC oxidase 1, three key enzyme genes for JA and ethylene biosynthesis which were critical forA. alternataresistance; 2)feruloyl-CoA 6’-hydroxylase 1(NaF6’H1), the key enzyme gene for phytoalexins againstA. alternata, scopoletin and scopolin; and 3) threeA. alternataresistance genes, long non-coding RNA (LncRNA L2),NADPH oxidase(NaRboh D) andberberine bridge-like(NaBBL28). SilencingLncRNA L2reducedA. alternata-induced levels of JA andNaF6’H1expression.NaRboh D-silenced plants were strongly impaired in ROS production and stomata closure responses.NaBBL28was the firstA. alternataresistance 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 againstA. alternatainN. attenuataby regulating different signaling pathways and defense metabolites. For the first time, such an important WRKY was identified inNicotianaspecies, providing new insight into defense mechanism ofNicotianaplants toA. alternata.